Nanomedicines for Improved Management of Ectopic Pregnancy: A Narrative Review.

Serum human chorionic gonadotropin (hCG) is a universal marker for early pregnancy detection in the clinic and can be used to detect EP since both intrauterine and ectopic pregnancies have actively expanding trophoblast tissue expressing hCG. While a single hCG measurement is useful for confirming pregnancy, it is well established that this is insufficient for differentiating a viable intrauterine pregnancy (IUP) from EP. Instead, the ratio of hCG at 48h compared to the first measurement at presentation is more predictive of the location and viability of the implantation. [ 15 ] In the case of a viable IUP, hCG levels are expected to rise a minimum of 35% in 2 days compared to the first test result at presentation, suggesting a predictive ratio of >0.74. [ 16 , 17 ] However, studies of hCG ratios have significant heterogeneity in specificity for EP, ranging from 28-98% depending on ratio cutoffs and whether hCG is increasing or decreasing. [ 15 ] The growing trophoblast forms a significant part of the placenta and initializes contact with the maternal circulation. Therefore, biomarkers of angiogenesis could potentially predict early EP and PUL. Vascular endothelial [ 18 - 20 ] and placental-like growth factors [ 21 , 22 ] have shown high specificity and sensitivity in detecting EP compared to viable and abnormal IUPs, but require further confirmation of their robustness. Markers of corpus luteum function, including progesterone and inhibin A, show conflicting results in differentiating EP from failing IUPs. [ 23 - 25 ] Other biomarkers associated with IUP viability, but which have irreproducible study outcomes for EP/PUL confirmation, include pregnancy-associated plasma protein-A2 (PAPP-A2), pregnancy-specific beta glycoprotein 1, and A disintegrin and metalloproteinase-12 (ADAM-12). [ 26 ] The development of biomarker-based sensors for EP and PUL detection requires an accounting of both temporal and pathophysiological circumstances since developing pregnancy and comorbidities can alter levels of serum marker concentration. Therefore, each marker must be highly sensitive and specific for EP/PUL and should be validated for different time points in the pregnancy development continuum. A review of the relevant literature suggests that nanomaterial-based enhancement of biomarker detection could increase the specificity and sensitivity for EP/PUL diagnosis. For example, one study sought to improve hCG detection sensitivity by exploiting a magnetic particle-linked monoclonal anti-hCG antibody. Another experimental immunomagnetic reduction (IMR) assay developed in 2015, using magnetic iron oxide (Fe 3 O 4 ) nanoparticles biofunctionalized with anti-hCGβ antibodies, demonstrated an LOD in urine samples at 0.36 mIU/mL, well below that of commercially available immunochromatographic assays (25 mIU/mL). [ 27 ] A study from 2019 reported that a carboxyl-graphene oxide-based surface plasmon resonance (SPR) aptasensor allowed ultra-sensitive detection of hCG in clinical serum samples. [ 28 ] This study proposed an aptasensor with high sensitivity, affinity, and selectivity to hCG, capable of detection well below normal physiological hCG levels. Another group created a carboxyl-molybdenum disulfide (MoS2) nanocomposite SPR biosensor for measuring PAPP-A2. [ 29 ] The experimental results demonstrated that the carboxyl-MoS2 SPR biosensor had high affinity and specificity for PAPP-A2 detection due to the proposed technology's unique electrical and optical properties, including a ~3-fold increase in SPR angle response compared to traditional SPR chips. PAPP-A2 protein can be utilized as a biomarker for clinical screening of down syndrome in utero and, together with PAPP-A, could predict adverse pregnancy outcomes. [ 30 , 31 ] In another study, utilizing a microbubbling assay, the authors reported an ultra-sensitive approach for hCG level detection that was ~150x more sensitive than current point-of-care hCG assays used in the clinic. [ 32 ] While these novel nanomaterial-based assays may demonstrate notable decreases in the LOD of hCG, and the authors claim that such approaches may enable more definitive diagnosis of early pregnancy, their usefulness in diagnosing EP at such an early stage is questionable, as such low levels of hCG are usually only expressed within the first week of pregnancy, presumably before the patient would suspect pregnancy or suffer any symptoms of EP. In the context of EP, however, there is a greater clinical need for assays paired with tissue-specific markers or imaging modalities to improve localization of the implantation site. In addition to protein and peptide biomarkers, nucleic acids such as circulating DNA, mRNA, and non-coding RNAs could be useful as predictive indicators for EP/PUL diagnosis. In fact, more than thirty placental micro-RNAs (miRNA) have been proposed as potential biomarkers for detecting EP and PUL. [ 33 , 34 ] For instance, increased levels of miR-323-3p were detected in the serum of women with symptomatic PUL who ultimately were diagnosed with EP, compared to women with viable IUP and abnormal IUP, with 37% sensitivity at a predefined specificity of 90% for EP. [ 35 ] The authors note that the relatively low sensitivity observed for miR-323-3p as a single marker is of limited clinical value. However, a combined panel of miR-323-3p, hCG, and progesterone increased the sensitivity to 77.8%, and a stepwise panel incorporating the three markers further increased sensitivity to 96.3% at the same fixed 90% specificity. Evaluation of hCG or progesterone as single markers, and a combined hCG + progesterone panel, at the same predefined specificity of 90%, revealed sensitivities of 14.8%, 3.7%, and 33.3%, respectively. [ 35 ] No nanotechnological tools are currently available for quantifying nucleic acid biomarkers for EP/PUL diagnosis. However, DNA-functionalized nanoparticles have been designed as probes to capture other miRNA markers using surface-enhanced Raman spectroscopy to diagnose preeclampsia and other pregnancy complications. [ 36 ] Collectively, basic science and translational studies have identified multiple potential biomarkers that can be further functionalized using nanomaterial-based methods to provide greater specificity and sensitivity for the early detection of EP and PUL. Direct visualization of a developing pregnancy is the most effective method of differentiating intrauterine from ectopic implantation. Consequently, transvaginal ultrasound (TV-US) imaging is used extensively in early pregnancy management with high sensitivity and specificity to enable less-invasive diagnoses of normal intrauterine pregnancy, early pregnancy loss, and ectopic pregnancy, as the gestational sac can typically be identified within the endometrial cavity using US as early as 2-4 mm or equivalent to 30-35 days or 4-5 weeks’ gestation, with other normal growth landmarks including the yolk sac and the embryo visible as early as 5 and 6 weeks, respectively. [ 37 ] TV-US has become the preferred imaging modality for diagnosis of pregnancy localization as, rather than relying on the inability to visualize an intrauterine implantation when using transabdominal ultrasound (TA-US), it relies on the ability to visualize an ectopic pregnancy, and does so with pronounced sensitivity (87.0-99.0%) and specificity (94.0-99.9%). [ 38 ] In addition to the differentiation of pregnancy localization, US imaging can also facilitate clinical assessment of placental development including size, location, adherence, and umbilical cord insertion at later gestations. [ 39 ] Interpretation of conventional gray-scale ultrasound relies on the echogenicity of tissues, depending on tissue density, with fluid-filled voids shown as black background. Contrast enhanced ultrasound (CEUS) can improve the signal-to-background ratio of conventional gray-scale ultrasound through the use of contrast agents, most commonly gas-filled microbubbles typically in the sub-micron size range. Several microbubble formulations have been approved for use in humans and have been shown to be safe and effective in both pregnant animal models and pregnant human patient cohorts for assessing placental function and development. [ 40 , 41 ] Various reports evaluating the efficacy of microbubble ultrasound contrast agents attribute their favorable echogenicity and backscatter coefficients, in part, to larger particle size and resulting increased gas volume. [ 42 - 44 ] Consequently, it may be difficult to imagine that the use of much smaller nanomaterial-based platforms would improve upon the currently available microbubble formulations. However, a review of the primary literature reveals a number of sub-micron formulations of various compositions, both targeted and non-targeted, that have been evaluated for their use as ultrasound contrast agents in vitro, and in vivo, with promising results. For example, a 2014 study by Wang, et al. investigated the use of perfluoropropane (C3F8) encapsulated within poly(lactic-co-glycolic acid) (PLGA) nanoparticles (hydrodynamic size (HDS):152 nm, polydispersity index (PDI): 0.221) as a US contrast agent, both in vitro and in vivo. Therein, the authors demonstrate increased US signal intensity in degassed deionized water (DI H 2 O) using B-mode imaging of the C3F8-PLGA nanoparticles at 13 and 22 MHz. Increasing concentrations of C3F8-PLGA nanoparticles resulted in a dose-dependent increase in US signal intensity that eventually began to diminish after 60 seconds. The authors also demonstrated an increase in US signal intensity of rat testicle following injection of C3F8-PLGA. [ 45 ] A 2013 study using gold nanoparticle-coated, perfluorohexane-encapsulated, PEGylated mesoporous silica nanocapsules (MSNC@Au-PFH-PEG, or “MAPP”) (HDS: 250 nm; PDI: unreported) revealed increased US signal intensity in degassed DI H 2 O, and in New Zealand white rabbits bearing VX2 liver xenografts and hepatocellular carcinoma tumor-bearing nude mice following intratumoural injection. [ 46 ] Additionally, the authors demonstrated enhanced high intensity focused ultrasound (HIFU) thermal ablation of ex vivo bovine liver and in vivo rabbit VX2 xenograft tumors using MAPP. A 2020 study by Dongying, et al. evaluated the use of both naked and glypican-3 (GPC-3) targeted gold nanoparticles (AuNP & GPC3@AuNP) (HDS: 195 & 230 nm, respectively) as exogenous US contrast agents in vitro, and reported a significant increase in signal backscatter compared to a solid, pure agarose gel as control. [ 47 ] Enhancement of US contrast using exogenous nano-size formulations can also be achieved by omitting fluorocarbons and encapsulating air, as evidenced by a 2016 report from Yildirim, et al. [ 48 ] In this study, the authors prepared mesoporous silica nanoparticles (HDS: 187-222 nm) (PDI: unreported) functionalized with octyl groups on the silica surface and solubilized using an amphiphilic copolymer surfactant (Pluronic F127), and encapsulating air as a US contrast agent. Therein the authors report that, following HIFU exposure, entrapped air within the hydrophobic capped pores of the nanoparticles is released to form larger US-responsive microbubbles. This study presents an intriguing solution to the extravasation constraints of larger microbubble formulations and the lower acoustic scattering cross-sections of much smaller nanoparticles. Furthermore, by encapsulating air rather than perfluorocarbons, the nanoparticles exhibited extended shelf-life on the order of 4+ months when solubilized and stored in PBS, or longer when lyophilized and subsequently resuspended. Taken together, these studies and others represent encouraging advances in the field of nanomaterial-based exogenous US contrast agents that could potentially be applied in diagnostic US imaging of EP. Photoacoustic (PA) imaging, also known as optoacoustic (OA) imaging, is a related hybrid imaging modality that combines the high contrast of optical imaging with the spatial resolution of ultrasound imaging at increasing tissue depth. PA imaging is made possible by the photoacoustic effect, where absorption of nonionizing electromagnetic energy in the form of radiofrequency or optical waves by photoabsorbent molecules causes transient thermoelastic expansion, generating acoustic waves that are detected by an ultrasound transducer array to produce images. [ 49 ] PA imaging exploits the optical absorption of several endogenous photoabsorbent molecules, such as oxygenated and deoxygenated hemoglobin, melanin, lipids, and water to construct 2D and 3D images of a tissue of interest. PA imaging has been used pre-clinically to monitor placental oxygenation and placental and embryonic function in pregnant rodent models [ 50 - 53 ] and a 2020 study by Maneas, et al., employed PA imaging to post-operatively validate laser-induced photocoagulation of anastomoses in twin-twin transfusion syndrome (TTTS) using ex vivo human placenta, further suggesting potential for the use of this imaging modality in obstetrics. [ 54 ] The use of exogenous contrast agents can further enhance the performance of PA imaging, and many exogenous PA contrast agents, including nanomaterial-based formulations, have been used extensively to image solid tumors. [ 55 ] It has previously been shown that employing exogenous contrast agents which absorb in near-infrared (NIR) window I (650-950nm), and especially NIR-II (1000-1700nm), can significantly enhance the signal-to-noise ratio of PA imaging due to reduced absorption by blood and water in this region of the electromagnetic spectrum. [ 56 , 57 ] Drawing on their previous work with nanomaterial-based fluorescence and photoacoustic imaging contrast agents for visualizing solid tumors [ 58 ] and endometriotic lesions, [ 59 ] Moses et. al., employed a poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether (PEG-PCL) nanoparticle encapsulating a hydrophobic NIR-I dye (silicon naphthalocyanine (SiNc)) (NIR-NP) as an exogenous contrast agent for enhanced photoacoustic imaging of the developing placentae of pregnant mice, with the aim of improving the detection of ectopic pregnancies ( Figure 2 ). [ 60 ] Therein they report that 24 h incubation of immortalized human placental trophoblasts with NIR-NP at a concentration of 25 μg mL −1 SiNc is sufficient to allow photoacoustic visualization of placental cells. Following intravenous administration in pregnant dams at gestational day (Gd) 12.5, the distinct PA signature of NIR-NP was observed in the placenta at 24h post-injection, contrasted with a complete absence of signal in the fetus, facilitating enhanced visualization of the placenta while avoiding any putative risk of direct fetal toxicity ( Figure 2C ). The inability of NIR-NP to traverse the placental barrier to the fetus is especially promising for diagnosis as, in a clinical scenario, if a pregnancy were determined to be a viable intrauterine implantation, the nanoparticle would theoretically pose no direct harm to the developing fetus. In fact, when mice were allowed to continue gestation following administration of NIR-NP, the pups were delivered without incident, no deaths were recorded, and no obvious signs of toxicity in pups or dams were observed over the course of 14 days post-parturition. These data demonstrating PA signal in the placenta, and absence of signal in the fetal compartments, are a promising step toward advancing the use of nanomaterial-based exogenous contrast agents for diagnostic PA imaging of EP. Fluorescence imaging is widely used in preclinical studies to obtain information about tissues both at macro and micro levels of observation, using either whole body in vivo imaging or fluorescence microscopy. [ 61 ] While fluorescence imaging possesses many favorable characteristics such as high spatial resolution, sensitivity, and ease of quantitative measurement, there are certain intrinsic limitations that still hamper its utility, including photon scattering, tissue absorption, and autofluorescence. Consequently, even when using exogenous small molecule fluorophores, in vivo whole body fluorescence imaging is limited to a depth of ~1 cm, beyond which photons encounter so many scattering events that the signal is rendered significantly diffuse. [ 62 ] It has previously been suggested that fluorescence imaging in the NIR region of the electromagnetic spectrum, especially NIR-II, could reduce tissue-associated signal attenuation, increasing the imaging depth to several centimeters, [ 63 ] and many subsequent studies have validated this hypothesis in human patients and numerous animal models. [ 64 ] In a recently published report, Moses et. al., demonstrated that intravenous injection of polymeric nanoparticles (NIR-NP) allowed visualization of the developing placenta through the intact dermis using in vivo whole-body fluorescence imaging in the NIR-I window, suggesting that nanoparticle-mediated fluorescence imaging may complement US and PA imaging when differentiating intrauterine and ectopic implantations. [ 60 ] Zhang et al. also demonstrated in vivo whole-body fluorescence imaging of gravid uterus following intravenous administration of lipid-polymer nanoparticles encapsulating indocyanine green (ICG). [ 65 ] However, while this method may be effective for pre-clinical visualization of implantation sites in small animals such as mice, this modality may ultimately not be suitable for diagnosing EP in human patients due to the limited depth of penetration of in vivo fluorescence imaging. While ultrasound imaging remains the preferred first-line imaging modality for investigating pregnancy, its efficacy is still largely operator-dependent and limited by interference from bowel gas, in addition to being restricted to a relatively narrow field of view. The use of magnetic resonance imaging (MRI) overcomes these limitations and is generally considered a safe nonionizing alternative to CT or X-ray but is still discouraged in pregnant patients unless the potential benefits outweigh the risks. This is especially so with regard to contrast-enhanced MRI, as gadolinium readily penetrates the placental barrier to the fetus and has been shown to retard development of rat and rabbit fetuses when administered at 2x and 2.4x, respectively, the recommended dose for humans. [ 66 ] Even without the use of exogenous contrast agents, MRI has excellent soft tissue contrast and is sensitive to blood, allowing diagnosis of hemoperitoneum that may indicate a ruptured EP, and assessment of the degree of hemorrhage. [ 67 ] For these reasons, MRI is often used in planning surgical approaches for treatment of EP and, while not a first-line approach for imaging pregnancies, MRI often reveals intrauterine and ectopic implantations when being used to investigate other concomitant pathologies. MRI is also used as a secondary imaging approach or problem-solving tool when US findings are inconclusive or in patients with non-ideal body habitus. [ 68 ] While contrast enhancement may improve the efficacy of MRI, as previously mentioned, the use of gadolinium-based contrast agents is discouraged in pregnant patients due to concerns of putative fetal toxicity. However, the development of nanomaterial-based contrast agents may alleviate these concerns, as demonstrated in a 2016 study by Shetty et al., which showed that a PEGylated liposomal gadolinium formulation prevented transport of gadolinium across the placental barrier following intravenous administration in pregnant mice at Gd16.5, which is roughly equivalent to the early part of the third trimester in humans. [ 69 ] Subsequent ICP analysis of gadolinium levels in fetal tissue revealed accumulation three orders of magnitude below that of placental tissue, and well below the detection limit, whereas administration of a commercially available gadolinium chelate (Multihance ® ) resulted in essentially equal levels of accumulation in fetal and placental tissues (within one order of magnitude). A 2019 study from Badachhape et al., further validated the inability of liposomal-Gd to cross the placental barrier to the developing fetus when estimating placental fractional blood volume in mice using T 1 -weighted MRI. [ 70 ] Ferumoxytol, an iron oxide nanoparticle formulation originally developed to treat anemia caused by chronic kidney disease, has since been repurposed for use as an MRI contrast agent as it was shown to shorten T 1 , T 2 , and T 2 * relaxation times and has a circulating half-life of 14-15 hours. [ 71 ] A 2020 study by Nguyen, et al., evaluated the effects of ferumoxytol on the maternal-fetal interface in pregnant rhesus macaques. [ 72 ] Following ferumoxytol administration via intravenous catheter and subsequent MR imaging of pregnant monkeys at Gd ~100, equivalent to the end of the second trimester of human pregnancy, no significant differences in iron concentrations were observed in fetal tissues or the maternal fetal interface when compared to controls, suggesting that ferumoxytol does not traverse the placenta to the fetus. A similar observation was noted in Prussian blue staining, and significant differences in iron concentrations were only observed in the maternal liver. Evaluation of progesterone, estrone, and estradiol levels in maternal plasma samples likewise revealed no significant differences in placental hormone levels following administration of ferumoxytol and MRI, indicating that the nanoparticles and imaging had no effect on placental endocrine function. Taken together, these data and that of numerous other studies [ 73 ] make a compelling case for the use of contrast-enhanced MRI as a first-line imaging modality to diagnose not only EP, but also other placental abnormalities including those that fall in the placental adhesion disorder spectrum – also known as morbidly adherent placenta. Careful consideration of the physicochemical properties of various nanomaterials and their resulting interactions at the maternal-fetal interface will hopefully drive the development of safe and effective nanomaterial-based exogenous contrast agents that can be used for contrast-enhanced MRI during pregnancy.

When EP is diagnosed, expectant management may be indicated in the confirmed absence of an intrauterine implantation and if the patient’s initial hCG value is low (<1000 – 2000 mIU/mL) and decreasing over time. [ 5 ] Expectant management is a “watch-and-wait” strategy where hCG levels are monitored longitudinally to determine if the pregnancy resolves spontaneously. When hCG values exceed this range and the EP is visualized, the patient is hemodynamically stable without evidence of intraperitoneal bleeding, and the gestation is early enough that hCG is <5000 mIU/mL and there is no fetal cardiac activity, medical treatment with MTX is indicated. Numerous international professional organizations in the field of obstetrics and gynecology recommend intramuscular administration of either a single, double, or multiple-dose regimen of MTX as a first-line medical treatment for ectopic pregnancy, but single-dose treatment is the most common regimen. [ 5 ] When a patient’s hCG levels exceed a certain threshhold (> 5000 mIU/mL depending on institutional guidelines) and TV-US reveals an adnexal mass greater than 35 mm or fetal cardiac activity, surgical management via laparoscopy or laparotomy is indicated. [ 5 ] For patients lacking a healthy contralateral fallopian tube who wish to preserve fertility, expulsion of the products of conception via salpingostomy is recommended. For patients with a healthy contralateral fallopian tube and those who have no desire to preserve fertility, salpingectomy (complete excision of the affected fallopian tube) is recommended. [ 5 ] Recent advances in experimental nanomaterial-based delivery systems have demonstrated significant potential to prevent systemic toxicities and off-target side effects of many common drugs including chemotherapeutics [ 11 ] and may provide alternative treatment strategies to mechanically destructive surgical procedures. While still a quite nascent field of inquiry, the use of nanomaterials in obstetrics holds great potential, although there are significant obstacles to gaining FDA approval for the use of any drug during pregnancy, including the reluctance of pregnant volunteers to enroll in clinical trials over fetal safety concerns, and pharmaceutical industry concerns of market restriction and legal liability. [ 74 ] Consequently, experimental use of nanomedicines during pregnancy has thus far been restricted to animal models. In the case of nanomaterial-based diagnostic and therapeutic modalities specifically for EP, experimental in vivo research also is restricted to pregnant animals with eutopic implantations because, while abdominal pregnancies have frequently been reported in various animals, and a handful of tubal pregnancies have been described in rhesus macaques, no reliable animal model of a true tubal pregnancy has yet been established. In a 2018 study, inspired by the binding of Plasmodium falciparum -infected erythrocytes to chondroitin sulfate A (CSA) expressed on the surface of trophoblasts during placental malaria, Zhang et al. evaluated the targeted delivery of MTX to trophoblasts of pregnant mice using lipid-polymer nanoparticles functionalized with a synthetic CSA-binding peptide (plCSA-MNP). [ 65 ] Their results indicate that, following intravenous administration, plCSA-MNP bind specifically to placental trophoblasts and not to other placental cell types. Moreover, no binding was observed in other maternal or fetal tissues that harbor CSA-expressing cells, and immunohistochemical staining revealed a similar distribution of nanoparticles within villous syncytiotrophoblasts of human placental explants incubated with biotin-conjugated plCSA-MNP. In comparison to free MTX, non-targeted nanoparticles encapsulating MTX (MNP), and MNP decorated with a scrambled peptide sham targeting moiety (SCR-MNP), all of which exerted only a slight effect on placental blood sinusoid volume, plCSA-MNP significantly inhibited fetal development and increased fetal demise ( Figure 3 ) when administered in a multiple-dose regimen (starting at Gd 6.5, every second day) at the same concentration of MTX (1 mg kg −1 ). HPLC analysis of MTX concentrations in fetal and placental tissues also showed a marked increase in placental MTX accumulation in the plCSA-MNP group compared to free MTX, MNP, and SCR-MNP. Apoptosis quantification in placental tissues using a TUNEL assay revealed a dramatic increase in the number of apoptotic cells in placentae of mice treated with plCSA-MNP when compared to free MTX, MNP, and SCR-MNP. Unlike free MTX, which induced both renal and hepatic toxicity, the livers and kidneys of mice treated with plCSA-MNP were comparable in appearance to those of control mice administered PBS. A 2013 study by Kaitu’u-Lino et al. investigated the feasibility of using EnGeneIc delivery vehicles (EDV) encapsulating doxorubicin to treat ectopic pregnancy. [ 75 ] EDVs are bacteria-derived nanobodies that can be used to encapsulate chemotherapeutics and other small molecules, and were previously used in a phase I human clinical trial to determine their safety and recommended phase II dose in treatment of recurrent glioblastoma when loaded with doxorubicin. [ 76 ] The authors noted that the human placenta expresses extremely high levels of epidermal growth factor receptor (EGFR) in comparison to other tissues, which inspired them to use a monoclonal anti-EGFR antibody as an active targeting moiety to deliver EDV-encapsulated doxorubicin specifically to placental trophoblasts. When human placental explants were incubated with EDVs encapsulating doxorubicin and targeted to EGFR ( EGFR EDV DOX ), doxorubicin was observed in the syncytiotrophoblast layer as early as 7 h post-treatment, compared to no observable accumulation in explants incubated with EDVs encapsulating doxorubicin and modified with an irrelevant antibody control ( GP120 EDV DOX ). By 24 h post-treatment, some accumulation of doxorubicin was observed in explants treated with GP120 EDV DOX , but to a lesser extent than those treated with EGFR EDV DOX , while no accumulation was noted in explants treated with free doxorubicin. Immunohistochemistry of placental tissue thin sections revealed an increase in apoptotic syncytiotrophoblasts following treatment with EGFR EDV DOX , compared to GP120 EDV DOX , when stained using M30, an antibody which binds a neoepitope of cytokeratin 18 produced by caspase cleavage during apoptosis. The authors noted that the extent of apoptosis observed in GP120 EDV DOX -treated tissues is consistent with the basal level of syncytiotrophoblast sloughing that is observed in placental tissues grown in standard mammalian cell culture systems. The two studies referenced here constitute the entirety of literature to-date dedicated to investigating nanomaterial-encapsulated chemotherapeutics specifically for the treatment of EP and suggest that the use of nanomaterials could potentially improve both the efficacy and safety of traditional chemotherapeutics when treating EP in human patients. Hyperthermal therapy, or simply increasing the temperature of a region of the body to achieve a therapeutic effect, has been used since antiquity for a variety of ailments. In the context of oncology, hyperthermia can be used as an adjuvant to traditional chemotherapy and radiotherapy regimens to produce synergistic effects by further sensitizing cancer cells to the aforementioned treatments. [ 77 ] A variety of methods can be used to produce both general and localized hyperthermia, including high-intensity focused ultrasound (HIFU) [ 78 ] and the application of alternating magnetic fields (AMF) to tissues harboring magnetic nanoparticles (magnetic hyperthermia). [ 79 ] The Australian veterinarian Marshall J. Edwards is widely credited with the discovery of maternal hyperthermia as a teratogen in humans. His pioneering rodent experiments demonstrated a correlation between the timing, duration, and intensity of maternal hyperthermic insults and the type of resulting fetal injuries, ranging from neural tube defects (NTD) and behavioral abnormalities to fetal resorption and abortion. [ 80 ] Experimental results indicated that prolonged exposure (~1 h) to temperature elevations ≥ 2˚C above normal biological temperature increases both the incidence and severity of birth defects, regardless of species. Numerous prospective and retrospective human studies subsequently confirmed Edwards’ experimental observations suggesting that maternal hyperthermia can cause NTDs. [ 81 - 88 ] Clinical studies have shown that the application of HIFU combined with suction curettage can be used for less invasive treatment of cesarean scar pregnancies (CSP) while simultaneously reducing the risk of hemorrhage and preserving future fertility. [ 89 ] A 2022 study by Moses et al. investigated the use of nanoparticle-mediated, NIR light-induced photohyperthermia for the potential treatment of EP in a pregnant mouse model. [ 60 ] Pregnant mice were intravenously administered photoactivatable polymeric NIR nanoparticles (NIR-NP) at Gd10.5, by which time maternal blood flow into the placenta has generally been established ( Figure 4 ). 24 h following injection, individual implantation sites in the uterus were clearly visible using both photoacoustic imaging and whole-body fluorescence imaging, and selected fetoplacental units were illuminated through the intact dermis using focused 780 nm light (0.9 W cm −2 ) via fiber optic cable for 10 min. Pregnant mice were then allowed to continue gestation for 6 days, at which time they were euthanized and the uteri examined for signs of resorbed pregnancies. Several underdeveloped implantations were observed in the NIR light-treated region of the uterus, accompanied by well-developed adjacent pregnancies within the same uterine horn. In contrast, the uteri of pregnant mice administered NIR-NP without NIR light exposure, and mice administered saline and exposed to NIR light, contained only healthy, well-developed placentae and fetuses. A detailed histological assessment of the treated fetoplacental units using H&E staining and TUNEL assay revealed viable uterine tissues (endometrium, myometrium, perimetrium) and placental spongiotrophoblasts, contrasted with infarcted, non-viable junctional zone (the interface between the placental labyrinth and decidua) and only necrotic fetal remnants in place of a healthy developing fetus. These results suggest that nanoparticle-mediated hyperthermia could be an effective alternative to chemotherapy and mechanically destructive surgical methods currently used to treat ectopic implantations, especially those of the tubal variety, and may consequently help to preserve patients’ future fertility when attempting to conceive following treatment for EP.

Ectopic pregnancy is a common disorder that affects a significant proportion of the global population, and its occurrence has only continued to increase over the last 50 years. As the leading cause of maternal death due to hemorrhage during the first trimester of pregnancy, early and accurate diagnosis is crucial for preserving the life of the patient and their future fertility, should they subsequently wish to conceive again. In this review, we discuss how nanomaterial-mediated diagnostic and treatment strategies, while still only theoretic for the diagnosis and treatment of EP, hold great potential to improve the current state of EP management by increasing the sensitivity of biomolecular assays; enhancing common clinical imaging modalities; improving the placenta-specific delivery of chemotherapeutic agents; and introducing new non-invasive methods to induce fetal demise. In the absence of a suitable animal model of EP, several biologically relevant replacements, each with their respective advantages and disadvantages, have been used to evaluate the biodistribution of various nanomaterial formulations and their interactions with the fetal-maternal interface, including pregnant rodents and monkeys, in vitro systems, human ex vivo placental explants, and “placenta-on-a-chip” models. [ 90 , 91 ] While these results suggest that nanomaterial-based therapeutic modalities possess great potential for improving current treatments and developing new alternative non-invasive treatments for EP that are promising for clinical translation, application of these modalities in a true animal model of tubal pregnancy is necessary to advance these and similar technologies to clinical implementation.

Ectopic pregnancy (EP) is a potentially life-threatening condition where a fertilized oocyte fails to descend the fallopian tube and/or implant properly in the endometrial lining of the uterus. The vast majority of human ectopic implantations (~97%) occur in the ampullar region of the fallopian tube, but are often observed in other locations including the ovary, the cervix, the abdomen, cesarean scars, and other more proximal or distal regions of the fallopian tube. [ 1 ] It is estimated that 1% - 2% of all pregnancies in the United States are ectopic implantations. [ 2 ] EP is also a significant contributor to total healthcare costs, as evidenced by a retrospective study that estimated the total annual economic burden of EP in the United States to be nearly $1.1 billion. [ 3 ] With the exceedingly rare exception of an abdominal pregnancy being delivered via laparotomy, all ectopic implantations are non-viable and put the patient at serious risk of tubal rupture, hemorrhage, hypovolemic shock, and even death. In fact, EP has historically been, and remains, the leading cause of maternal death due to hemorrhage in the first trimester of pregnancy in both developed and developing countries. [ 4 ] As such, timely diagnosis of EP is crucial for reducing the risk of tubal rupture and subsequent bleeding. Current diagnostic methods for suspected EP include serial measurement of human chorionic gonadotropin (hCG) and ultrasound (US) imaging, with correlation to clinical history. Rising or falling hCG patterns are helpful for guiding early pregnancy management, but EP can mimic a failing intrauterine implantation (miscarriage) or even a very early normal intrauterine pregnancy. [ 5 ] While US imaging is non-invasive and extremely effective at diagnosing EP with high sensitivity and specificity, its success is largely dependent on the skill of the operator, the size of the gestational tissue, and the patient’s body habitus, and diagnosis may still be erroneous in up to 40% of cases. [ 6 ] However, visualization of an ectopic implantation is still the most effective diagnostic method for EP. Current management strategies for suspected or confirmed EP include expectant management, medical treatment with methotrexate (MTX), or surgical expulsion of the products of conception or complete excision of the affected fallopian tube, with each strategy having its respective advantages and disadvantages based on the clinical situation. Ectopic pregnancies that fail to spontaneously resolve may eventually lead to rupture and hemorrhage during expectant management. MTX is mostly effective at treating ectopic pregnancies, but common adverse effects include alopecia (0.5-10%), photosensitivity (3-10%), rash (0.2-10%), nausea and vomiting (10%), thrombocytopenia (10%), as well as fatigue, malaise, and shivering. More rarely, serious adverse effects occur, including stomatitis, hepatotoxicity, and pneumonitis (1%). [ 7 - 9 ] Critically, ongoing EP may ultimately result in tubal rupture, and expose the patient to serious risk of hemorrhage and death. Surgical intervention is surely the most effective method of treating EP, but carries its own risks of intra- and post-operative complications. [ 10 ] Therefore, although significant advances have been made in the management of EP, there is still an urgent need for improvement in terms of early, accurate diagnosis and medical and surgical treatments that minimally impact patients‘ quality of life and future fertility. Nanomedicine possesses great potential to improve the current state of diagnostic and therapeutic strategies for EP, as nanomaterials of various composition have long been praised for their ability to encapsulate various hydrophilic and hydrophobic molecules including small molecule drugs, imaging contrast agents, and nucleic acids; prevent their cargo from degradation; extend their time in systemic circulation; and deliver greater amounts of these cargoes to a specific tissue of interest. [ 11 ] By functionalizing the surface of nanoparticles with active targeting moieties, even greater accumulation of their encapsulated payloads in targeted tissue can be achieved while simultaneously decreasing their accumulation in off-target sites, thereby reducing the severity of systemic side effects of various drugs including chemotherapeutics. [ 12 ] The placenta is a highly vascularized organ and a promising target for nanomaterial-mediated systemic delivery of imaging and therapeutic agents. Recently, the pre-clinical use of nanomedicines during pregnancy has been explored for the treatment of various obstetric and pregnancy complications including pre-eclampsia, fetal growth restriction, gestational trophoblast disease, choriocarcinoma, etc. [ 13 , 14 ] While the aforementioned reviews highlight several promising advances in these directions, there is a relative scarcity of reports relating to the use of nanomaterials specifically for the diagnosis and treatment of EP. Current diagnostic and treatment strategies ( Figure 1 ) and their advantages and disadvantages are summarized and, where available, reports of nanomaterial-mediated modifications to those methods and newly proposed strategies are evaluated.