Perspectives on the Technological Aspects and Biomedical Applications of Virus‐Like Particles/Nanoparticles in Reproductive Biology: Insights on the Medicinal and Toxicological Outlook

Abstract

Increasing data on the infection indicate that maternal infections are severe. Under the realms of vaccine development, virus‐like particles (VLP)/nanoparticles (NPs) hold the promise of targeted control of therapeutics transfer across the placental barrier with the potential to trigger innate immune responses. Though the placenta is known to act as a barrier against exogenous materials, viruses exploit the transport systems and overcome the barrier properties. VLPs can be strategically designed to obtain the necessary mechanisms for navigation across the placenta and immune response. However, several knowledge gaps on the chemical, viral transmission strategies and the host defense response exist owing to the highly dynamic etiology of the placental barrier. This further complicates the toxicological analysis of the developed therapeutics. Herein, placental physiology and functions are discussed in significance with chemical toxicology, viral infections, and the host defense. Further, the promising applications of VLPs and perspective on their design to overcome the placental gatekeeper to gain the necessary immune response or therapy are provided. Finally, a holistic approach to various bioengineering models for studying chemical toxicants, viral infections, and effects of VLPs is provided to facilitate better translation of these VLPs to clinical applications.

Introduction

The placenta is a vital organ in pregnancy as it regulates fetal and maternal health by performing the actions of the endocrine glands, liver, kidney, and lungs of the fetus. They further act as a barrier against external insults. Fetal and maternal coordination is achieved by several cells that form the placental barrier and receive signals from either side. Such signals ensure the proper growth of the fetus while effectively eliminating toxins and other waste. Several diseases associated with placental insufficiency or placental dysfunction can result from the improper function of the placental barrier. Owing to the extreme complexity of the gestational process, research efforts are continuing for several decades to decrypt the same. Studying physiology and pathological phenotype of the placenta poses a big bottleneck to pathologists owing to the multifactorial etiology involved in a particular condition in addition to the dynamic and rapid phenotypical change in the placental barrier throughout pregnancy. Though analysis of the structural and phenotypical characteristics of the placenta can provide the necessary insights into the function, the technical and ethical limitations make it difficult to obtain the entire quantitative description of the structural growth and function of the placenta.^{[ 1 ]} Recently, much interest has been shown by scientists in placental research, owing to its connection to birth mortality and health at later ages.^{[ 2 ]} However, it is a relatively understudied process within bioengineering aspects including physical properties of the tissues, blood, and oxygen transfer. In a similar vein, response to viral infections is another less explored part of placental function. As the placenta is not the primary host entry point for viral infection but is rather translocated by secondary transmissions, studying the repercussions of any viral infection is more complex than other common infectious processes owing to the dynamic in vivo functions of the barrier.

Though it is widely known that placental coordination is maintained by signals from both maternal and fetal circulation, an understanding of the detailed mechanisms is still elementary.^{[ 3 ]} Placental phenotypic changes are also regulated by these signals. For instance, insufficient vascular endothelial growth factor (VEGF) or placental growth factor in maternal circulation can cause poor endothelial regulation and placental dysfunction or insufficiency.^{[ 4 ]} These implications on the placenta are generally identified by placental observation after delivery.^{[ 3 ]} However, several pathological conditions with placental insufficiency/dysfunction are manifested at the early stages of placental development, which in turn trigger conditions like preeclampsia, placenta accreta, intrauterine growth restriction (IUGR), stillbirth, and several other maternal and neonatal complications.^{[ 5 ]} Moreover, viral infection during pregnancy causes several conditions including preterm birth, congenital malformations, and developmental problems, which in turn have long-term health implications for the newborns. Adverse outcomes including abortion, stillbirth, and congenital abnormalities have been recorded with some viral infections.^{[ 6 ]} Many of the viruses that are documented to affect the placenta have not been explored enough to understand the transmission mechanism. Recent evidence suggests that the function of the placental barrier is highly critical against several viruses including zika viruses (ZIKV), both severe acute respiratory syndrome coronavirus (SARS-CoV and SARS-CoV-2) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV).^{[ 7–9 ]} These findings highlight the placental host strategies and the virus programming to overcome the same and infect placental cells. A detailed review on the transmission of several classic and upcoming viruses resulting in severe maternal–fetal outcomes has been provided recently.^{[ 10 ]} Unfortunately, the exact mechanisms of viruses for immune invasion and transport across the placenta have not been identified. This has been attributed to the practical challenges in studying the placenta using conventional techniques. All over the world, recorded data have suggested the high susceptibility of pregnant women to viral infections including influenza, rubella virus, varicella, SARS-CoV, and MERS-CoV.^{[ 8,11 ]} It is now even more important to understand how the virus interacts with the host and its transmission across the placenta in the wake of the recent pandemic of SARS-CoV-2.

As maternal blood is the sole supplier of nutrients and oxygen to the fetus, exposure of the mother to drugs or pathogens can increase the translocation of exogenous substances across the placenta and have detrimental effects on the fetus. Hence, there is a continuous demand for more studies on virus transmission across the placenta and therapeutic solutions to overcome the same. Although advances in the field of clinical toxicology, medicine, and pharmaceutics can be considered advantageous, there prevails a level of skepticism toward medications for pregnant women and their effect on birth defects. With the success of tetanus vaccination during pregnancy, recommendations for immunization during gestation against several infectious diseases have increased. Considering the advances in targeted drug delivery using nanoparticles (NPs), focus on developing virus-like particles (VLP) or NPs is currently being explored by several research groups.^{[ 12 ]} Unlike conventional vaccines, VLPs are genetically engineered to offer greater immunogenicity and safety with the scope for application for other chronic diseases.^{[ 13 ]} Research efforts in recent years have thus broken the notion that the placenta is an impenetrable barrier, but rather a leaky barrier with highly selective transport mechanisms that can be exploited for therapeutic interventions.^{[ 14,15 ]} Thus, drawing inspiration from these viruses, nanocarriers/VLPs can be designed to specifically target several pathogenic mechanisms. However, an elaborate toxicological assessment of the developed VLPs is highly critical owing to the proven developmental toxicity of some exogenous substances from several epidemiological studies.^{[ 16 ]} In addition, the transplacental transfer of the VLPs, antibodies, and the effect of maternal health conditions on the transfer is needed.^{[ 17 ]}

While designing materials for overcoming the barrier defenses is a major challenge, testing of these developed materials is exceedingly complex as testing drugs on pregnant women raises several ethical issues.^{[ 18 ]} Modeling the placental barrier in vitro can be considered an alternative for studying the pathophysiology of the barrier at various stages of pregnancy. For instance, several in vivo, placental explants and in vitro models of the placental barrier have been studied by researchers with the prime focus of analyzing the substances that cross the barrier.^{[ 9,19–21 ]} However, models that mimic the pathophysiology of several placental disorders that are triggered by several factors including pathogenic transmission are highly scarce^{[ 22 ]} Hence, understanding the physiology of the placenta facilitates researchers to create a more efficient model that effectively replicates the transport and metabolic properties of the placenta. Also, most of the models fail to represent the dynamic phenotype of the placental barrier. This can be attributed to the poor knowledge of the phenotypical complexity involved in a particular condition that can change the pregnancy outcome.^{[ 23 ]} To our knowledge, there is no literature on models that effectively replicates the placental feature over the entire course of pregnancy.

In this review, the different approaches to studying placenta and virus/toxicant/VLP transmission across the placenta have been considered to provide a holistic framework. As VLP can be designed to have versatile applications including antigen presentation, drug delivery systems, or vaccine candidates, this review will specifically focus on VLP design strategies for placental transfer and innate immune response. To understand the strategies of virus pathways across the placenta, a brief and clear understanding of placental development and phenotypic features are necessary. Next, it is essential to know the specific pathological implications of viral infections. Finally, the bioengineering techniques to understand these physiological and pathological changes in addition to testing the nanomaterials/chemical toxicant translocation are focused on in the final parts of the review. Finally, advances in the state-of-art and future outlooks have been provided.

Placental Physiology and Transport Properties

Anatomically, the placental barrier is composed of multiple cells arranged in a multilayered pattern (Figure  1A), contributing to interface separating the maternal and fetal sides. The dynamic nature of the placental barrier is due to the specialized cell types, including subtypes of trophoblasts originating from the blastocyst trophectoderm precursors.^{[ 24 ]} Fundamentally, the placenta is composed of the trophoblast layer and an endothelial layer that actively separates the fetal flow from maternal blood. A major part of the barrier is contributed by a thin, multinucleated layer of syncytiotrophoblasts (SCT), formed by the fusion of villous cytotrophoblasts (VCT), alongside variable cytotrophoblast populations like extravillous trophoblasts (EVT) depending on the gestational age.^{[ 25 ]} The SCTs are directly in contact with maternal blood, while the vascular endothelial cells are part of the blood vessels within the chorionic villi containing fetal blood. In addition to acting as a site of exchange between the maternal and fetal organs, these SCTs have specific endocrine functions, including hormone production. Thus, the functionality of the placental barrier is determined by all these cells with SCT being a major regulator of barrier properties.^{[ 26 ]}

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Similar to intestinal villi, the placental villi act as the primary regulator of nutrient and matter exchange. Upon fertilization and blastocyst entry into the uterus, they divide into trophoblast and embryoblast. These trophoblasts invade the uterine lining and are further divided into SCT and VCT. These two cells are developed on either side of the villi. However, the trophectoderm layer is developed into the chorion layer of the fetus.^{[ 27 ]} Together, these components contribute to the fetal compartment of the placenta. During the progress of pregnancy, there is a slow reduction in the VCT from chorionic villi (Figure 1B,C). In addition, villi structures are formed around the same period to increase interaction between maternal blood and fetal vessels.^{[ 28 ]} With an increasing gestation period, several villi cell types, including immature intermediate villi, mesenchymal villi, mature intermediate villi, stem villi, and terminal villi, contribute to placental signaling (Table  1 ). The villi structure can either be floating or anchored (Figure 1). Floating villi are contributed by the VCT inner layer and covered by SCT and maternal blood, which in turn flows into the intervillous space (IVS). Usually, the placental phenotypic characteristics are determined at the end of the first trimester. The late second trimester and third trimester are the stages where the placenta works at full capacity. However, pathogenic mechanisms that start in the first trimester and early second trimester are yet to be explored.

Table 1 Villous development in placenta over pregnancy

Gestational age	Cell types	Properties
First trimester	Mesenchymal villi – extraembryonic mesenchymal cells invade the initial villi	Villi filled with these cells are more primitive, indicating poorly developed villi Formed at 12 days postconception Further gets differentiated into endothelial cells, macrophages, myofibroblasts blood cells, smooth muscle cells, and fibroblasts
Mid-first trimester	Stem villi	Connects the chorionic plate Can be identified as fibrous stroma with vessels of varying sizes Development can be detected with smooth muscle and central stromal fibrosis Negligible fetal maternal exchange
Mid-first trimester	Immature intermediate villi	Reticulate stroma containing macrophages called Hoffbauer cells and a discontinuous cytotrophoblast layer Growth centers of villous trees. Act as preliminary sites of signaling and exchange
Early-second trimester	Mature intermediate villi	Long and slender with surface ramifications. No fetal vessels Develop into terminal villi Critical for fetal–maternal exchange
Late-second and early-third trimester	Terminal villi	No stroma Predominant sinusoidal capillaries Contains discontinuous cytotrophoblasts Exchange of O2, CO2, and nutrients

Hofbauer (HB) cells are the fetal macrophages that can be detected during the early gestational period. They are responsible for villous remodeling, immune response, and hormone secretions. These cells are particularly important while studying the immune response to viral infections. For instance, evidence suggests that zika virus (ZIKV) and human immunodeficiency virus (HIV) particles have been identified in HB cells when the first-trimester placental infection was studied.^{[ 29,30 ]} However, the ability of these HB cells to either act as a reservoir or as limiting barriers to viral replication remains unknown. Scientists have identified secreted proinflammatory cytokines in response to several infections during in vitro culture of cells.^{[ 31 ]} However, in vitro models used in these studies do not entirely replicate the in vivo complexity, as the VCT and SCT microenvironment influence the responses of HB cells.

Similarly, the decidua is contributed by the specialized endometrium of the implantation site and also provides immunotolerant functions in fetal trophoblasts. This immune privilege of the decidua is by its cell composition with the strict regulation of chemokine expression.^{[ 15 ]} This decidua is a multicellular structure with VCT and maternal cells. The decidua basalis contains 40% immune cells; out of those, 70% are decidual natural killer (dNK) cells, 25% are macrophages, and the remaining are T-cells.^{[ 15 ]}

Understanding transport restrictions and access routes for solutes across the placenta is crucial in understanding the placental barrier mechanism. Transport across several barriers of human physiology is crucial for any diagnostic and therapeutic development including toxicological assessment.^{[ 32,33 ]} Most solute transfer is known to begin in the later developmental stages of the fetus when the placental structure is mature enough to withstand fluid diffusion.^{[ 14 ]} In this sense, if a mother is exposed to an exogenous substance during early gestational ages, translocation across the placenta in the early trimester could result in 20% of postnatal malformations. Though a complete understanding of the inner and highly selective mechanisms regulating the transport of fetal metabolites across the placenta has not yet been derived, scientists have attained a general idea of these transport pathways. Like any other biological transport system, the nutrient diffusion across the placental barrier is mediated and governed by the concentration gradient, extent, and thickness of the placental barrier, that in turn is determined by the paracellular permeability and electrical potential difference.^{[ 34 ]} Three mechanisms of exchange have been reported across the placenta: transporter-mediated mechanism, diffusion, and endocytosis/exocytosis (Figure  2 ).

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Simple diffusion occurs passively without ATP expenditures through the layers of cellular membranes and the cytoplasm. Small hydrophobic molecules cross the membrane easily by the concentration gradient and the rate of diffusion is governed by Fick's law 1 $\text{Rate}=\frac{\text{Surface\hspace{0.17em}area\hspace{0.17em}}\times \text{Concentration\hspace{0.17em}gradient\hspace{0.17em}}\times \text{Krog}{\mathrm{h}}^{\prime }\text{s\hspace{0.17em}Contant}}{\text{Thickness\hspace{0.17em}of\hspace{0.17em}the\hspace{0.17em}membrane}}$

The main factor that influences the gradient is the maternal and fetal circulation that constantly refreshes the reservoirs. Changes in the maternal and/or fetal blood flow can have a profound effect on the net flux. Thus, the transport of lipophilic compounds and gases by diffusion is considered flow dependent.^{[ 35 ]} However, in the case of limiting conditions such as high-altitude pregnancy, the adaptive responses of the placental barrier can result in changes in the barrier thickness or surface area to facilitate stable exchange.^{[ 35 ]}

Several substances including small molecules, antibodies, and drugs are generally transported by protein receptors across the placental barrier. Such receptor-regulated transport is bidirectional, making communications between the fetal and maternal organs indispensable. The apical side is contributed by the maternal compartment with high transport proteins that act as both influx and efflux transporters.^{[ 36 ]} Even with the lack of microvilli, the basal side possesses many receptors for the effective exchange of nutrients (Figure 2). Any changes to the expression and availability of these receptor proteins triggered by a viral infection or drug exposure could result in detrimental effects on fetal health. For example, alterations in the gene expression contributing to varying levels of fatty acid, amino acid, and glucose transporter have been proven to be a contributing factor in IUGR.^{[ 3 ]}

The primary barrier that arbitrates the transport of components across the placenta is the SCT. However, other cells play a supporting role in the proper barrier function and placental transport. Several reports stress the importance of placental transporters in fetal health. For instance, GLUT1 is the principal glucose transport receptor among the GLUT-transported family, involved in transport across trophoblasts. Similarly, lipid transport across the placenta is mediated by scavenger receptors that bind to low-density lipoproteins and high-density lipoproteins, which have been reported. The expression of these receptors increases across gestational age. However, it was identified that their expression is suppressed at term in pre-eclampsia pregnancy with severe growth restrictions.^{[ 37,38 ]} Such data demonstrate that changes in the expression of receptors could have adverse effects. Unfortunately, these innate metabolisms and transport pathways are hijacked by drug molecules and other foreign substances like viruses, NPs, etc.,^{[ 33 ]} thereby resulting in undesirable placental or fetal outcomes. While exposure to drugs can be controlled, viral transmission after infection of the host is a highly unpredictable interaction sequence and can result in adverse effects.^{[ 6 ]} Studying the virus transmission and the host cell response can throw more light on placental defense mechanisms as well as strategies to prevent or treat viral transmissions during pregnancy.

Placenta as a Barrier for Viral Infection

During the overall gestation period, the placenta acts as the primary defense against heterogeneous transmission of chemicals, toxicants, and viruses and provides an infection-free milieu for the uterine environment. Immune responses to pathogens are downregulated in the placenta to a certain degree to ensure proper function. Thus placenta can be considered a site of immune privilege.^{[ 39 ]} Fine-tuned and highly regulated balance between immune activation and antigen tolerance for the embryo is needed for successful pregnancy. A detailed review of the immunological aspects of healthy pregnancy and in pathological condition has been provided in the study by Yang et al.^{[ 40 ]} Despite the elementary defenses of the placental barrier, viruses are known to be transmitted from the maternal circulation with the placenta as the portal for entry. This has been attributed to the immunotolerant environment that is adapted during the pregnancy to ensure the appropriate function of the placenta.^{[ 6,40 ]} Similar to other pathogenic processes, the exact mechanism of the shielding effect against viruses and the viral strategies to bypass this shield remains unknown. Viral transmission in utero can occur by several transit routes, that is, 1) maternal endothelium to extravillous cytotrophoblasts, 2) transmission of infected macrophage from maternal blood to trophoblasts, 3) vertical transmission (through urogenital tract), and 4) paracellular transmission into fetal capillaries.^{[ 41 ]}

From placental physiology, it can be understood that SCT acts as the first line of defense against any pathogenic attack. The physical nature of the highly fused SCT provides a powerful physical barrier for microbial transmission. As many microbes are programmed to bypass cellular barriers by weakening the cell junctions, SCT has evolved to possess a defensive barrier characterized by the presence of a dense actin network and by the complete lack of cell junctions.^{[ 22 ]} SCT has been recorded to exhibit antimicrobial activity against infections from coxsackieviruses (CVB), poliovirus (PV), vesicular stomatitis virus (VSV), human immunomodulatory virus (HIV), cytomegalovirus (CMV), vaccinia virus (VV), and herpes simplex virus (HSV). However, cytotrophoblasts are known to be susceptible to infections by the aforementioned viruses.^{[ 11 ]} The barrier also synthesizes high nitric oxide synthase, microvillus associated glycosaminoglycans, and secret antiviral interferons (IFN).^{[ 42 ]} Thus, virus infections at the early stages of gestation could result in adverse effects, as the placental anatomy is not equipped to display antimicrobial properties at earlier stages. As mentioned previously, VCT differentiates into SCT via two pathways involving floating villi and anchoring villi. As differentiation into EVT in anchoring villi invades uterine endometrium, there is high susceptibility to viral vertical transmission. The EVTs reach the maternal decidua by invasion, where it becomes a part of the multicellular phenotype, thereby serving as replication sites for vertical transmission.^{[ 15 ]}

Recent studies have also confirmed the vertical transmission of zika virus (ZIKV). ZIKV replication is enabled in the placenta by receptors expressed, during trophoblast differentiation (Figure  3 ).^{[ 43 ]} While multiple receptors like TIM-1, AXL, and Tyro3 have been shown to facilitate ZIKV entry, the precise viral entry receptors remain unknown.^{[ 43,44 ]} ZIKV is hypothesized to seed the fetal compartment by invading the EVTs and paraplacental route through amniochorion.^{[ 28 ]} Only minimal receptors for viral on the SCT have been described. For example, surface receptors like NRP2 and PDGFRA are expressed in less quantity in SCT (Figure 3). Alternatively, the neonatal Fc receptor (FcRn), which is expressed on the apical side of SCT, can be hijacked by viruses like ZIKV, HIV, and CMV. Similarly, Transferrin receptor 1 (TfR1) for iron transport which is expressed on SCT has been exploited by the hepatitis C virus, suggesting a possible viral transport mechanism.^{[ 45 ]} Currently, the exact transmission route of the virus across the placenta is poorly defined. The viral replication in the fetal capillaries provided an entirely new transcriptional profile unlike those in adult infections, which can be attributed to the downregulation of genes responsible for cytokines regulation and fetal sensory development.^{[ 46,47 ]} Such a response was identified by characterization of viral antigens of rubella virus in the chorionic villi, within cytotrophoblasts and endothelial cells. For instance, chemokine-expressing CCL14 and CCr1 gene is generally highly expressed in early pregnancy by human endometrium. Rubella virus infection is downregulated in virus-infected fetal endothelial cells, unlike adult cells by 2.31-fold and by 5.70-fold, respectively.^{[ 47 ]} Some virus infections like HSV are known to spread from basal decidua and replicate via IVS. Similarly, studies on the SARS-CoV-2 have also documented viral transmission across the placental barrier. This was confirmed by the high expression of angiotensin-converting enzyme 2 (ACE2), the primary receptor of the SARS-CoV and SARS-CoV-2, in SCT, VCT, vascular smooth muscle cells, and fetal endothelial cells.^{[ 7,8 ]} Debelanko and team performed a clinicopathological analysis of the SARS-CoV-2-infected placental sample and identified that SCT damage was accompanied by fibrin deposition, with zero SARS-CoV-2 positive neonates.^{[ 7 ]} In line with this, a systematic review was done on the vertical transmission of SARS-CoV-2, and the results indicated a very low probability of vertical transmission with the available data.^{[ 48 ]} Hence, these detrimental pathologies of viral infections need to be understood in detail to devise strategies for virus-mediated fetal congenital conditions.

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Albeit the lack of evidence on the exact mechanism, these results indicate the ability of viruses to develop strategies to invade placental cells.^{[ 49–51 ]} The current methods of determining viral infections during pregnancy rely on serology to measure immunoglobulin levels and viral detection by identifying viral nucleic acids.^{[ 6 ]} However, better testing methods to avoid higher risk and better outcomes are in demand. The existing literature on the ability of some viruses to overcome the defense of the placental barrier and affect fetal development calls for research methods for identifying the underlying transmission mechanism. Research groups are currently focusing on developing model systems, mimicking placental barrier, to gain insight into these mechanisms.^{[ 9,31,51,52 ]} However, each study has reported different routes of transmissions across the placental barrier. Such a diverse set of findings can be attributed to the model systems used for identifying the mechanism and thus enunciate the highly adaptable strategies that virus particles undertake to the crossplacental barrier. Further results on virus−host interaction at the placenta are crucial for the future unfolding of novel vaccine-based therapies against viral infections.

Placental Host Defense Strategies

While the capacity of some viruses to cross the placental barrier has been demonstrated in the previous sections, the placenta continues to host a series of defensive mechanisms that protect the fetus from a wide range of pathogenic organisms. Understanding viral–host interactions necessitates an understanding of the placenta's general defense mechanisms. This will further enable the development of strategies to fight against new viruses in the future. The placenta, which is in direct contact with the decidual lining of the uterus, provides the initial immune defense against several viruses. The decidual layers possess NK cells, macrophages, neutrophils, and innate lymphoid cells that contribute to the immunological defense mechanisms^{[ 22 ]} (Figure  4 ). The placenta exhibits a defensive mechanism by secreting antimicrobial components that target a wide range of viruses. For instance, Fung and his team identified a novel IFN type I (IFN-ε), in the female mice reproductive tract which is also expressed in human endometrium, thereby inducing canonical antimicrobial pattern-recognition receptor (PRR) pathways.^{[ 53 ]} The PRR pathways are specifically encoded to recognize conserved pathogen-associated molecular patterns (PAMPS). Antiviral signal induction and regulation of PRR is a common defense strategy adapted by placental cells, with the expression primarily regulated by the age of gestation and cell type.^{[ 11 ]} Unlike the findings by Fung and team, research by Delorme-Axford et al. failed to identify a pre-existing IFN response in human trophoblasts.^{[ 54 ]} Racicot and colleagues’ recent study with mouse models indicated the importance of IFN signaling in viral transmission, and inhibition of placental IFN affects placental function and immune regulation.^{[ 55 ]} However, it remains to be determined whether the human placenta can possess similar defenses against viruses based on the studies using mice models. Data on shielding effects from viral infection by IFN secretion have been contradictory,^{[ 11 ]} thus providing a paradigm for future researchers to understand the immunological responses by the reproductive system and host defense mechanisms.

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An alternative mechanism involving the expression of neonatal Fc receptor (FcRn) on the SCT surface for specific transport of IgG has been reported (Figure 4). This receptor has been known to be exploited by viruses including ZIKV, HIV, and CMV to overcome the barrier defenses.^{[ 56 ]} Similarly, transferrin receptor 1(TfR1), expressed on the surface of SCT for iron transport to the basal side, is exploited by the Hepatitis C virus for entry across the placental barrier.^{[ 57 ]}

PRR pathways were encoded to recognize a wide range of pathogens including bacterial lipopolysaccharide, lipoteichoic acids, DNA, RNA, glucans, and peptidoglycans. These are critical for both innate immune response and toll-like receptor (TLR) activation of antigen-presenting cells (APCs). This is accomplished by upregulating the major histocompatibility complex (MHC) and stimulatory molecules.^{[ 58 ]} TLRs are commonly studied PRRs for several viral infections and their expression depends on several factors including pathogen exposure, cytokines, gestational period of viral exposure, and previous viral infections.^{[ 12 ]} Further, there is also a variation in the expression pattern with different cell lineages, although the reason behind the same is unclear. It could be possible that minor alterations in the cytokine profiles change the microenvironment, which in turn changes as pregnancy proceeds.^{[ 59 ]}

In addition to the secreted antiviral components, the placenta further produces several proteins and peptide compounds with antiviral properties. These components are released into the amniotic fluid as a part of the defense strategy. For instance, antimicrobial peptides can directly kill bacteria, yeast, fungi, and viruses and further regulate host immune response by recruiting myeloid cells and lymphocytes to the infection site, thereby mediating TLR activation.^{[ 60 ]} SCT secretes high levels of β-defensin and their subtypes in response to potential pathogenic infection in maternal blood. In addition to this, several types of antiviral IFNs are secreted as the initial defense mechanism against viral infections. Specific IFNs carry out mechanisms to either attach to receptors and activate PAMP pathways or restrict viral entry into host cells by other routes. However, studies suggest that improper regulation of IFN secretion could result in other pathological conditions by affecting the syncytin-mediated fusion of VCT.^{[ 61 ]}

Maternal immunization provides defense strategies against congenital transmission of viruses and their seeding in the placenta, an opportunity currently being explored against CMV^{[ 62,63 ]} and HSV.^{[ 64,65 ]} Generally, maternal immunoglobulin F (IgG) is transported around 13 weeks of gestation, and as a result, a fetus that is exposed to pathogens after 12 weeks shows fewer congenital defects.^{[ 52 ]} This transfer of IgG is a pH-dependent process, where a portion of IgG is attached to the neonatal Fc receptor (FcRn) in SCT cells of the placenta. This transport mechanism is unique and has recently gained much significance after its identification. Thus, it was noted that such a receptor immune interaction was not available in other cells, including fetal endothelium due to nil expression of the receptor FcRn by the other cells of the placenta. Similarly, noncanonical Fcγ receptors like FcγRI, FcγRII, and FcγRIII are known to be expressed in placental cells and could have a combinational mechanism of immune transfer.^{[ 66 ]} Such a unique biological system requires further exploration of the underlying mechanism for the future development of immunization strategies.

However, considering the urgency in developing safe immunization strategies for pregnant women, the development of solutions that utilize the same strategy as the IgG transfer process could speed up the research on the safe antiviral delivery to fetus across the placenta. For instance, molecular dynamics (MD) applications can be adapted to develop scaffolds containing both antivirals and interaction sequences of IgG with FcRn.^{[ 67 ]} It was suggested that IgG is transferred to SCT via phagocytosis into endosomes. This is generally mediated by the membrane-bound FcRn.^{[ 68 ]} The interaction of IgG and FcRn occurs via basic amino acid residues H310 in the heavy CH₂ domain and H435 and H436 domain in the Fc region. Using MD simulations, peptide sequences can be designed that react with FcRn and such peptides can then be prepared using vector expression to be covalently attached to nanocarriers.

Inspired from Virus: Design of Virus-like Particles/NPs

Virus-like particles (VLPs) or NPs, are supramolecular structures that exhibit close resemblance to viruses but do not possess the infectious ability. These are nanoscale structures that are self-assembled by viral structural proteins produced in a variety of systems including bacteria, plants, animals, and insects. VLP/NP can be tailormade to target specific cells of the immune system and also present antigens to the immune system, thus making them ideal vaccine candidates. The several classes of VLP-based vaccine design for different applications have been reviewed in detail by Koudelka et al. (2015).^{[ 69 ]} Structural proteins from HIV, hepatitis B, and C and bacteriophages have been commonly used to produce VLPs that are usually in the size range of 20–200 nm.^{[ 70 ]} VLP from Hepatitis virus is one of the widely explored candidates. Owing to their size and shape, they elicit the immune responses of the actual virus without replication in the target cells. Two types of VLPs can be developed from viruses by either using the core antigen or the surface antigen. The core antigen VLP assembles to form icosahedral geometry and can be produced using cell-free protein synthesis or E. coli cytosolic accumulation.^{[ 71 ]} Generally, the geometry of VLPs falls into two types (i.e.,), icosahedral for spherical VLPs and helical symmetry resulting in nanorod formation.^{[ 72 ]} The different types of VLPs along with their properties and production have been reviewed in detail in the study by Rohovie et al.^{[ 71 ]}. Although many traditional vaccines have been found effective, several inherent risks including unstable toxicity and mutation possibility make it difficult to guarantee the longevity of conventional vaccines. These risks can be eliminated by developing VLPs with greater immunogenicity and safety. VLP are made of copies of viral proteins with self-assembling properties. Such a design could theoretically represent a safe vaccine or drug delivery candidate against pathogens while retaining the ability to overcome placental defenses similar to original viruses. Several techniques can be used to tailor and design VLPs including genetic engineering, biomineralization, bioconjugation, encapsulation, and infusion.^{[ 69 ]} Detailed analysis of the commonly used VLPs and their production methods have been reviewed in several articles along with a critical appraisal of the upcoming design strategies.^{[ 69,71–73 ]} VLPs can serve versatile applications as they are expected to be ideal vaccine candidates, carriers of adjuvants or antigens to stimulate immune response, and also in antigen representation. As VLPs/NP can be produced in different shapes, conformations, and using several components, they have found applications in a variety of fields including catalysis, energy, biomedicine, photonics, etc.^{[ 72 ]} A recent review discusses the progress in the versatility of these VLPs.^{[ 13 ]} Antigen representation in a repetitive array is expected to develop stronger innate immunity when compared with individual soluble antigens. These VLPs are also expected to enhance the antigen uptake by APCs. The structure of VLPs can be modified based on the intended application to display foreign antigens for broader immunity and can further be repurposed as nanocarriers for drug/antigen delivery for prophylactic effects.^{[ 13 ]} NPs are known to cross the placental barrier by the paracellular passage which is further governed by size, shape, charge, and hydrophilicity.^{[ 74,75 ]} Thus, it is equally important to study these aspects of the VLPs to predict their passage through the barrier. The application of VLP as vaccines can further be extended by a modification to the structure to express adjunct proteins of other viruses/pathogens.^{[ 76 ]} This can be achieved by the fusion of compounds with membrane antigens or by endogenous expression.^{[ 77 ]} From an immunity point of view, VLPs represent PAMP due to their structural conformation similar to viruses. This can result in adaptive immunity due to discrimination by placental host defense strategies.^{[ 78 ]}

For conventional VLP, virus capsid proteins are developed initially by expression in an appropriate protein expression system.^{[ 79 ]} For example, VLP-based vaccine system for ZIKV has been proposed by the baculovirus expression of premembrane (prM) and envelope (E) proteins that could be self-assembled into ZIKV-like particles. Such ZIKV VLP has been tested to stimulate high antibody titers in mice.^{[ 80 ]} Alternatively, the capsid protein core of the VLP can be replaced by NPs like gold NPs.^{[ 76,81,82 ]} Second, fusion proteins can be designed and conjugated with the VLP core by incorporating immunorelevant exogenous epitopes.^{[ 79 ]} However, care should be taken during the design of the epitope to exhibit stable structures. It is noteworthy to mention that some VLPs can elicit a better and stronger immune response. For instance, the hepatitis B virus (HBV) core antigen HBcAg undergoes dimerization to form spike-like structures on the HBV surface.^{[ 83 ]} This strategy can be effectively replicated in VLP synthesis to increase their immunogenicity. The same epitope was more effective when displayed on influenza A antigen-containing VLPs, in which case neutralizing antibodies were formed.^{[ 84 ]} This is an indication of how crucial it is to select the VLP. To select the right VLP for treatment, one must match the targeted disease with the selected VLP. Often, this involves testing several combinations to determine which produces the best immune response. Thrane and team developed VLP-based vaccine for delivering mSA-VAR2CSA antigen for placental malaria caused by Plasmodium falciparum.^{[ 85 ]} The VLP assembly was modified to induce an optimal long-lasting immune response, by adding a biotin acceptor site (AviTag) to the coat protein. Such an assembly enables the anchoring of monovalent streptavidin (mSA)-fused protein to the biotin without compromising the VLP assembly. VLPs are highly advantageous as their surface components can be modified to couple with foreign antigens. For example, VLPs from the RNA bacteriophage AP205 can simultaneously display two antigens for placental malaria (VAR2CSA) and HPV L2 RG1 for human papillomavirus

Polysaccharides represent an important class of compounds for antibody targets against several bacterial infections. Though they do not affect inducing helper T cells by themselves, they are often conjugated with other protein antigens for developing conjugate vaccines. For instance, multimerization of protein–saccharide conjugate indicated an increased antibody production against Shigella infection.^{[ 86 ]} Thus, it is crucial to activate the innate immune response of the system rather than delivering the antigen alone. For this purpose, vaccines are usually given with an adjuvant.^{[ 87 ]} In addition to the adjuvant presence, other factors including the internal and external features of the VLP influence the quality of the immune response. TLR activation can provide the innate immune response to an infection by packaging o bacterial RNA into the VLP.^{[ 88 ]} The same can be further extended for pandemically relevant viruses as well. Coronavirus VLP can be produced by the expression of membrane, envelope, and S proteins via coinfections of recombinant baculoviruses.^{[ 89 ]} Such Coronavirus VLP was found to increase the expression of costimulatory molecules like several interleukins and IFN. VLPs can be specifically designed to have varying effects based on the route of administration. For instance, mice immunized with SARS-CoV VLPs via the intraperitoneal route exhibited cellular and humoral immune responses. However VLPs administered via intranasal route result in lower IgG.^{[ 90 ]} Such versatility can be exploited to locally target the therapeutic response. Thus, based on the individual coronaviruses, these VLPs can be developed specifically to counter any future pandemics. For instance, wild-type SARS-CoV-2 and group-specific antigen (Gag) VLPs share a diameter of 145 nm approximately, thus making them highly promising candidates for the generation of new vaccine candidates for coronavirus.^{[ 91 ]} Similarly, a novel VLP-based immunization for Zika virus has been reported recently by Garg and team^{[ 92 ]} and was found to possess neutralizing antibodies postimmunization. However, the studies were conducted in nonpregnant mice and no data on the VLP impact on the pregnancy were reported.

It is crucial to consider the type of pathogen targeted and the immune response required to design smart VLPs. For instance, in case of intracellular pathogens, VLPs targeting the dendritic cells with antigen crosspresentation can be considered an ideal choice for design.^{[ 79 ]} One primary advantage of VLP-based strategies for infectious disease is their immunogenic properties like traditional vaccines, but without the viral genome, thus making them safe. In case of vaccines as attenuated pathogens, there is a possibility that they can replicate in the host cell and spread in the population or recombine resulting in new variants.^{[ 79 ]} While most of the research on VLP-based therapy is still evolving with only limited candidates in the clinical trial stages, their effect on pregnancy and placenta continues to be a mystery. It is primarily recommended that antenatal care providers should take an active part in counseling the pregnant population on the benefits of immunization during pregnancy and also the risks associated with it. Furthermore, it is imperative to have more research on the effects of these VLP-based immunizations on pregnancy to enable a better understanding of the outcomes.^{[ 93 ]}

Initially, VLPs were developed as a solution to contain the disease by the corresponding native virus. However, continuous technological evolution and the maneuvering possibilities with VLP have opened up new avenues in their application including the treatment of chronic diseases and cancer. Vaccines are biological response modifiers that have the potential for tumor tolerance. Thus, using VLP potential, molecules including short peptides can be delivered to dendritic cells to induce cellular or humoral response.^{[ 94 ]} For instance, numerous VLP-based vaccines against human papillomavirus (HPV) are currently being investigated for breast cancer, melanoma, and leukemia.^{[ 95 ]} VLPs are high-precision materials that can be designed to exhibit the desired size, shape, application, targeted delivery, and immune response in addition to wide disease specificity. It has been recently proven that not only the size of the particles can be controlled, but geometry as well.^{[ 69 ]} Further, there is huge scope of VLP commercialization using plant viruses and bacteriophages on a large scale by molecular farming in plants or bacteria. However, their environmental safety needs to be considered by heterologous expression of genome-free VLPs. While VLP design by itself is a huge challenge, testing the same for maternal complications comes with challenges that are mostly ethical. In such cases, in vitro and in vivo models hold the key to the effective clinical translation of this promising drug delivery/vaccine platform.

Research Models for Placental Physiology, Pathology, and Barrier Characterization Studies

As the study of the placenta is hampered by ethical issues, alternatives involving animal and in vitro cell models are widely used to understand placental changes toward viral infections. Further, replicating the placental barrier is crucial to assess the effectiveness of pharmaceutics against viruses. While animal models provide a wholesome approach to pathological and drug translocations studies, the spatiotemporal and immunological differences in the placenta fail to necessarily replicate the actual human placental responses. It is important to understand the existing model systems and their shortcomings to determine the appropriate route toward developing effective pharmaceutics. Based on the study requirements, a befitting model can be chosen.

In Vivo and Ex Vivo Models

Several pregnant animal models are often used to study the placental membrane. Mice are the most popular animal models for studying placental responses to viral infections. In addition to their low cost and breeding time, mice are the widely preferred models as they recapitulate several aspects of human disease including after responses to viral infections.^{[ 52 ]} Mouse models, particularly, knock-out mice, pose another advantage of studying individual genes and their effect on placental function during infections. This possibility was found to be critical while studying ZIKV infection in mice. For instance, wild type mice failed to show any indication of ZIKV replication inside the placenta owing to IFN signaling pathways. To dampen this innate immune response against ZIKV infection IFN receptor, knock-out mice (Ifnar1 ^−/−) were used.^{[ 96 ]} However, a closer analysis of immunocompetent pregnant mouse models revealed viral particles translocation into the cerebellum and cortex of the fetus.^{[ 97 ]}

Trophoblast invasion is a diverse phenomenon among mammals, with particular regard to a number of cellular layers between fetal and maternal organs (Figure  5 ). In the case of lab-bred mice and other primates, a hemochorial (three-layered trophoblasts) placental type is established with trophoblast invasion through three layers of uterine epithelium, stroma, and maternal artery to get in direct contact with maternal circulation.^{[ 98 ]} In case of animal models, there is no perfect model that would exactly replicate the complex human placental architecture, even with hemochorial placental species like mice or nonhuman primates. In addition, from the vantage point of an obstetrician, some placental insufficiencies are found only in humans and possibly higher apes.^{[ 99 ]} Although such caveats on pregnant animal models exist, the majority of the knowledge on the early phenotypic abnormalities has been derived from these models. Further, differences in actual physiology, structure, and uterine environment along with the cost to conduct such studies handicap the application of such models (Table  2 ).

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Table 2 Merits and demerits of available experimental models of the placental barrier

	Model	Subtype	Specification	Advantages	Disadvantages
Ex vivo	Human palcental perfusion	Multicell type	Whole human placenta freshly obtained after caesarean section	No patient safety issues Best ethical route to study placental transfer Possible to study drug passage and placental metabolism Rate of drug transfer possible All compartments available for sampling (including placenta)	Isolated organ Absolute physiological state like in the whole body not attainable Viability of perfused tissue restricted Interindividual variance Use of reference substance required Difficult to extrapolate data to in vivo condition
	Nonhuman placental perfusion in vitro and in situ	Multicell type	Whole animal placenta freshly obtained	Drug passage and placental metabolism study possible Less interindividual variations in inbred animals Drug concentrations in correlation to time possible	Ethical considerations for animals used Data extrapolation to that of humans difficult Absolute physiological condition not achievable Interspecies variance Difficult to obtain large series of samples from one small animal in the in situ studies
In vivo	Murine	Multicell type	Experiment on alive mouse or rat	Cheaper Easy to use Longitudinal sample available	Anatomical differences Parturition begins differences Per pup small sample size Pup number large Endocrine differences
	Nonhuman primate	Multicell type	Experiment on alive nonhuman primates	Physiological similarities Parturition initiation similarities Longitudinal samples available Reliable Fetal number small	Expensive Difficult to handle
	Human	Multicell type	Experiment on alive human	Preferable primary source Ideal physiology and anatomy Standard parturition initiation	Difficult clinical trial Difficult samples acquisition Need for infrastructures to store samples In
	2D cell culture	Single-cell type	Primary cells	Human cells Physiological relevance In vivo characteristics maintained	Interindividual variability Difficult to culture Only a part of the whole tissue studied
In vitro	2D cell culture	Single-cell type	Immortalized cells	Derived from human cells	Need proof of physiological relevance
	Transwell coculture	Multicell type	Human cells	Sustainable culture	In vivo properties lost Only a part of the whole tissue studied
		Multicell type	iPSC	Sustainable culture Universal lab standard	Limited cell type Not optimized for the feto−maternal interface Only a part of the whole tissue studied
		Coculture	Immortalized cells	Able to study cell−cell or cell−collagen interactions Able to study signaling propagation and barrier function	Difficult to culture cells on both cells on both sides of the membrane Low throughput High signal-to-noise ratio Limited cell type Difficult to expand beyond two cell-type coculture
	3D cell culture	Spheroids	Primary cells	3D cell growth Maintains better in vivo properties High throughput Mixed coculture possible	Limited phenotypic assays with small cell number Time consuming Nonuniform Difficult to organize properly into a proper organ structure
	3D cell culture whole tissue	Cell sheets	Amniotic cells	Complete amnion layer Longer cultivation than amnion explants Easy to image	Very weak Only mimicking the amnion layer Nonuniform
		3D cell printing	BeWo trophoblasts and primary human placental endothelial cells	Multiple feto−maternal interface layers are possible Tissue organization is proper Uniform production	May cause unwanted shear stress to cells during printing Each cell type characterization is time-consuming For feto–maternal interface, ECMs have not been demonstrated yet
		Explant culture	Primary cells	Tissue organization is proper Mimics in vivo signaling	Sample acquisition is hard Need extra infrastructures to store samples properly Culturable for only up to 72 hours or less
	Placenta on chip	Perfusion system	Placental cotyledon	Various compartment model designs are possible Mimics the physiological process closely Possibility to study detailed physiological processes under a microscope Bioprinting of cells or scaffolds can be utilized to create coculture OoC devices	Multiple cell culture compartments need to be accessed by various fluids To incorporate physiologically relevant ECMs can be challenging Cannot completely mimic complex human placental system

Researchers have been unable to develop a practical solution to test the placental response to virus or xenobiotics exposure. For instance, scientists have failed to replicate placental implications for a broad range of pharmaceuticals, including testing of diabetic drugs or vaccines or medications for viral infections like HIV.^{[ 14 ]}

Placenta-on-Chip and Organoids

The challenges and limitations in studying the complex placental interface have spurred interdisciplinary advancements in cell culture to study the structure and function of such systems. Easy manipulation of placental cell lines and trophoblasts are some of the obvious merits of adapting a simple in vitro system. Replicating the native biology of the phenotypic systems can be done by several approaches like scaffolding the cells using hydrogels, organ on chip (OoC) with bioprinting approaches, and simple in vitro culture in transwell setup and organoids (Figure 5). In vitro models hold the advantage of high simplicity and easy setup. However, several in vitro placental models were developed with the primary focus on SCT cells without fetal endothelial cells.^{[ 100 ]} The use of transwell setups for trophoblast culture has been one of the most popular approaches for recreating the placental barrier. In this, the trophoblasts cells are cultured as monolayers into the Transwell setup to create the apical and basolateral side.^{[ 101 ]} Such a model system is efficient for studying the bidirectional transport of compounds across the placenta. These in vitro models have been used to study trophoblast physiology, immune aspects including infection, drug interaction, and nutrient uptake. Alternatively, the use of cocultures has been employed to tackle the simplicity of monolayer cultures. Coculture models still have drawbacks related to assigning the stage of pregnancy to the model.^{[ 102 ]} Also, the static nature of these systems fails to recapitulate the cellular complexity of the placental barrier (Table 2).

The demerits of in vitro and in vivo studies can be mitigated by placenta-on-chip technology. Using interdisciplinary approaches, the so-called OoC can provide compartmentalized structures for individual components of any biological system.^{[ 103 ]} Despite the advancements of OoC technology in several organs over the years, the field of obstetrics only just began applying this promising technology to study pregnancy and related complications.^{[ 21,104–107 ]} It is based on microfluidics that manipulates fluids in small channels. For a placenta-on-chip model, three components are mandatory: a maternal compartment with chorionic cells with maternal blood, a fetal compartment with fetal endothelial cells, and a basement membrane in between.^{[ 100 ]} Placenta-on-chip models use fluid shear stress that has been correlated with better microvilli formation unlike static in vitro cultures.^{[ 105 ]} Unlike other in vitro models, microfluidics offers the possibility of mimicking the early stages of fertilization, which is otherwise considered a black box of pregnancy. For instance, Gnecco and team developed a microengineered model of human endometrium for decidualization.^{[ 108 ]} Similarly, Zhou et al. modeled the peri-implantation phase of human pregnancy using extended blastocyst and stem cell-derived trophoblasts.^{[ 109 ]} Owing to their recent popularity in feto-maternal research, several models are being studied to answer various gaps in pregnancy-related pathology and therapy. Detailed reviews of these models and their application have been reviewed in detail.^{[ 3,100,110,111 ]} Considering the better replication of in vivo like features, placenta on chip is highly suitable for identifying the translocation of VLP/NP as well. While other Ooc models have been successfully employed to identify NP impact, placenta-on-chip models for VLP transport and effect are still in their infancy.

Considering the multifaceted role of the placental barrier, it is impossible to develop a single model that recapitulates all the features and functions. Thus, it is pertinent to develop specific models for addressing particular questions and then integrate the derived knowledge to understand the bigger picture underlying the complex mechanisms. For example, what are the molecular events that lead to congenital diseases from maternal infections? How are the immunological response and the placental barrier phenotype altered as a result of viral infection? Zhu et al. developed a microfluidic model of trophoblast and endothelial cells and infected them with E.coli bacteria to identify changes in barrier integrity and inflammatory cytokine secretion.^{[ 112 ]} While it is evident that determination of placental effects is possible using placenta on chip, the use of appropriate fetal membranes like amnion epithelial cells for amnion membrane OoC^{[ 111,113 ]} in parallel with placenta-on-chip can throw some light on the infection-induced stress on the fetal cells. Another significant approach to utilizing the full potential of placenta-on-chip models is to employ appropriate cell cultures unlike the immortalized or transformed cells widely used. As a result, increasing attention is being given to using induced pluripotent stem cells as a source of human reproductive cells.^{[ 110,114 ]} Alternatively, cells derived from an infected placenta can also be employed as an effective strategy to identify the pathological implications after maternal infection. Recent studies have also explored the feasibility of integrating bioanalytical tools with other OoC to rapidly analyze the inflammatory response.^{[ 115 ]} Such strategies can be effectively adapted for placenta-on-chip/ fetal membrane on chip to monitor the immune responses to viral infection. Within the domains of infectious diseases and toxicant transport across the placental barrier, it is important to create models that would be representative of key stages of gestation and also work as an effective barrier and appropriate immune response to infections.

Future Perspectives in Design Strategies of Virus-like Particles/NPs and Toxicity Testing

Improving maternal and fetal health against infection and its related congenital diseases has been a constant area of focus in several global health plans. Despite the availability of several immunization programs and schemes, one-third of the neonatal morbidity is attributed to the infections .^{[ 116 ]} Owing to the recent pandemic of SARS-CoV-2, there is an increasing momentum to design, develop, and implement immunization strategies for pregnant women.^{[ 117 ]} During gestation and postnatal period, antibodies from the mother are transferred to the child through the placenta or breast milk, respectively, as a natural process against several infections. However, viruses are constantly evolving to bypass host defenses while the host cells strive to evolve in response. While this fight toward the survival of the fittest has mostly resulted in checkmates by the host, there have always been consequences of birth defects in pregnant women before arriving at this point.

Nanotherapy against viral infections in conjunction with the placental barrier is considered to hold the answer to this intense battle owing to its highly versatile properties. NPs can be specifically designed to have adaptable surface properties that specifically target the placental barrier and/or in response to stimuli. Three major issues need to be addressed while considering VLP/NPs to cross the placental barrier, that is, the developmental phase of the placental barrier, the characteristic properties of NPs and nature, as well as factors related to virus need to be thoroughly studied. Pregnant women are more likely to contract infectious diseases due to adaptive physiological processes suppressing their immune systems during pregnancy.^{[ 118,119 ]} VLP or other nanotherapy may be considered, depending on the phase of pregnancy, as early pregnancy has a thick placental barrier protecting the developing fetus, whereas late pregnancy has a thin placental barrier providing better exchange of gases and nutrients. Because pregnancy causes significant physiological changes in women, they are a special population group. For example, depletion of plasma proteins increases the amount of unbound drugs in the systemic circulation in pregnant women.^{[ 120 ]} In addition, pregnancy induces an increase in circulating blood volume, cardiac output,^{[ 121 ]} increase in bioavailability due to prolonged intestinal transit time,^{[ 122 ]} and increase in glomerular filtration rate and alterations in the activity of metabolizing enzymes in the liver.^{[ 123 ]} This type of physiology in pregnant women aids in the understanding of NP uptake, clearance, and translocation at the human placenta.

Aside from factors such as gestational age, dose, dosage, route of administration, and drug clearance, the physicochemical properties of NPs such as chemical composition, size, shape, surface chemistry, porosity, and elasticity can influence NP biodistribution.^{[ 124 ]} The most obvious property influencing the transport is the size of VLP/NP, with smaller NP showing a higher transfer rate than larger VLP/NPs. However, there is no cut-off range on the size as the transport also depends on the type of material used.^{[ 125 ]} Due to the body's innate immune system, the reticuloendothelial system (RES) eliminates foreign bodies from circulation through immune cell phagocytosis and/or opsonization. This is why the surface properties of the VLP or NPs should be highly considered, as hydrophobic and charged particles undergo higher opsonization as compared with hydrophilic and uncharged particles. Despite the popular assumption that positively charged particles show higher translocation relative to negatively charged molecules, a clear trend in the size dependency was not identified when the effect of NP properties on the placental crossing was reviewed.^{[ 125 ]} Shape dependency on VLP translocation is another aspect that has recently gained significance. This is particularly of significance in the case of VLPs as they tend to have a shape similar to the virus which could increase their ability to cross the barrier. For instance, uptake of different geometries of silica-based NPs and fiber-shaped particles was studied and it was identified that the orientation of NP to cell surface influences the uptake.^{[ 126 ]}

A better understanding of viruses, their lifecycle, and physiological virus–host interaction mechanisms is required to develop an effective antiviral therapy that can be used prophylactically. Although vaccination has helped to confer immune protection against some diseases, this approach still has many challenges, such as the requirement of extensive time and resources, ineffectiveness against the mutant strain, and inability to combat viruses in already infected patients. The downside to traditional antiviral medications is that, despite their high effectiveness, they are difficult to use and have strong side effects.^{[ 127 ]} Fortunately, as nanotechnology advances, novel strategies for developing broad-spectrum nanotherapeutic platforms to combat viral infections may emerge.^{[ 128,129 ]} Viral spread can be limited by inhibiting the virus’ binding to host cell receptors or by preventing enveloped viruses from fusing with the host's membrane. Various antibodies or receptor-specific ligand conjugations of the NPs can be designed for such an approach. In multivalent drugs, monoclonal antibodies can be used to target a very specific epitope, whereas bispecific and trispecific antibodies can target multiple epitopes.^{[ 130 ]} Using antibodies as passive immunotherapy neutralizes pathogens directly by recognizing antigens present on the pathogen's surface that are responsible for binding to or allowing entry into host cells. Similarly, virus mimicking nanodecoys like VLPs can also be designed where the NPs are specifically modified to neutralize viruses via ligand−receptor interactions.^{[ 131 ]} One of the ways is that NPs are conjugated with ligands that selectively bind to viral receptors required for cell invasion. By catching and blocking these ligands, the nanodecoys prevent viruses from interacting with the intended target cells, and such inactivated viruses can later be cleared by the body´s immune system. The other way is that the receptor-binding domains of the virus can be isolated or synthesized and then conjugated with the NPs, which could then bind to the host receptors, preventing the binding of actual viruses. By regulating independently the valence and the spacing between ligands, it could be possible to design potent monovalent or polyvalent scaffolds as well as learn more about structure–activity relationships.^{[ 132 ]} Apart from that, a cell mimicking nanosponge can also be designed by wrapping the purified cell membrane of the host cell over the NPs. These nanosponges have been shown to be effective against many viruses^{[ 133 ]} including SARS-Cov-2, and have a promising role in combating various mutagenic variants.^{[ 134 ]} Working with such nanodecoys does not require biosafety level 4 like that for many virulent viruses and hence makes it more reliable to work in a normal laboratory setting. Viral envelope-disrupting structures such as AH or C5A peptides can be used to rupture small viruses of less than 70 nm in size. However, they are unable to rupture larger viruses. Many domains of NPs can incorporate such peptides, allowing even larger virus envelops to be disrupted.

While the development of VLPs and NP holds the potential for effective therapy during pregnancy, their translation to clinical practice needs to go through a long and laborious drill of toxicity testing. There are numerous probabilities for toxicity due to exposure to VLPs/NPs by pregnant women^{[ 135 ]} (Figure  6 ). A detailed review of these aspects has been provided in other studies.^{[ 16,136,137 ]} While in vitro models and other placenta-on-chip models could provide basic details on this aspect, complete knowledge of the translocation and uptake of existing NPs is still largely unknown. One way to work toward this would be to employ several toxicity applications like the use of machine learning (ML)^{[ 138,139 ]} and artificial intelligence (AI)^{[ 140,141 ]} to identify the structure-activity relationships. Data from these can be coupled with in vitro, continuous placenta-on-chip models and ex vivo models.^{[ 142,143 ]} Nonetheless, current knowledge on the placental transfer of exogenous substances (viruses, VLPs, NPs) and their toxicity is limited, necessitating additional research to broaden our understanding of these topics.

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Conclusion

As biomimetic and bioinspired NPs are expected to have diverse functionality, it holds the promise of control of their physicochemical attributes toward efficient immunization and therapeutics delivery for pregnant women, unlike conventional drug candidates. Despite the availability and approval of several VLPs for infectious diseases as immunization approaches, considerable translational barriers exist, particularly during gestational immunization for most of the developed VLPs. Transplacental transfer of VLPs from mother to fetus in the early epochs of life is one aspect that needs to be addressed during any future VLP development. By identifying the physicochemical and placental translocation effects of these VLPs for immunization, their application can be further extended for other therapeutics delivery.

In addition, VLP-based vaccines are expected to share similar problems encountered with regular vaccines in terms of the longevity of the host response. VLPs as vaccines for pregnant women will encounter problems with efficient immune response as the transport of these VLPs across the placenta remains unclear. Though they hold more potential than subunit vaccines due to their authentic confirmation, the toxicity of these VLPs, particularly those developed with nanomaterials, remains a major gridlock in this field of research. Another consideration is the translation of these materials from research tools to the clinical population, which is further complicated by the complexity of clinical trials. Because VLPs are generally administered to healthy populations, their application to pregnant women must address safety and tolerability concerns. To ensure the safety of mother and infant lives, additional deep and thorough toxicological studies, including genetic and epigenetic changes, inflammatory and oxidative stress effects, and placental and fetal changes should be prioritized. Further, the development of VLP for infection treatment should also take into account sex-specific placental function and effects. Thus, several questions regarding safety and efficacy need to be answered before these VLPs are strategically employed in clinical practice.

Acknowledgements

This work was partly supported by Hamad Medical Corporation Grant IRGC-05-SI-18-360.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

V.C., A.V.S., S.P.D., and R.S.M.: contributed in conceptualization. V.C., A.V.S., and R.S.M.: contributed in draft curation. V.C., A.V.S., R.S.M., and S.D.: contributed in draft preparation. V.C., A.V.S., R.S.M., S.B., S.P.D., S.D., P.L., S.S., and M.P.: contributed in review and editing. V.C. and R.S.M contributed in graphic design. A.V.S., S.P.D., P.L., and A.L.: contributed in supervision. A.V.S., S.P.D., S.B., and A.L.: contributed in funding acquisition.

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