Gold nanoparticle biodistribution in pregnant mice following intravenous administration varies with gestational age.

AuNPs of 15 nm diameter were synthesized using the Frens method as previously described [ 29 ]. Briefly, gold chloride dissolved in deionized water was stirred and heated until boiling. Sodium citrate was added, and the reaction observed for ~15 minutes until the solution became a deep red color. The resultant AuNPs were filtered to remove aggregates then resuspended in water. The AuNPs were passivated by adding 10 μM of 5 kDa methoxy-poly (ethylene glycol)-thiol (mPEG-SH) to the AuNP solution while stirring. After stirring for 1 hour, the PEG-coated AuNPs were purified by centrifuging (21,000g × 30 min) the sample to form a pellet and removing unbound mPEG-SH with the supernatant. The purified PEGylated AuNPs were dispersed in sterile phosphate buffered saline (PBS). Nanoshells with ~120 nm silica cores and ~15 nm thick gold shells were synthesized by the Oldenburg method [ 30 ]. First, 3–4 nm diameter gold colloid formed by the Duff method [ 31 ] were combined and reacted with aminated silica spheres (Nanocomposix) and rocked for several days at room temperature to form “seed”. Unreacted gold colloid was removed from the seed by centrifugation (2800 rpm, 25 min) and resuspended in water. The purified seed solution was then combined with a solution of potassium carbonate containing HAuCl 4 and formaldehyde to form complete gold shells. The synthesized silica core/gold shell NSs were purified by centrifugation (500g × 5 min, thrice) and resuspended in water, then 5 kDa mPEG-SH was added to the samples at a final concentration of 10 μM. After reacting overnight at 4°C, the PEG-coated NSs were purified by centrifugation to remove unbound mPEG-SH. Samples were diluted in PBS and stored at 4°C until use. Transmission electron microscopy (TEM) was used to visualize both AuNPs and NSs after synthesis. TEM samples were prepared by diluting AuNPs or NSs in water to an optical density (OD) of 0.3 or 1, respectively, at their peak plasmon resonance (520 nm for AuNPs; 800 nm for NSs) and placing a drop of sample on poly-l-lysine copper grids. Samples were imaged using a Zeiss Libra 120 Transmission Electron Microscope. The mean diameter of the AuNPs and NSs was determined by measuring the diameter of 20 particles manually in ImageJ. The extinction spectra (and OD) of the NPs were recorded using a Cary60 UV-visible spectrophotometer, using water as a baseline. For dynamic light scattering and zeta potential measurements, the AuNPs were diluted in water to OD 520nm =0.3 and the NSs were diluted in water to OD 800nm =1. Then, the samples were evaluated using an AntonPaar LiteSizer500 instrument. The reported hydrodynamic diameter and zeta potential are the mean and standard deviation of 20 sample measurements. All mice were maintained, bred, and used in accordance with Animal Use Protocols approved by the Institutional Animal Care and Use Committee at the University of Delaware (AUP #1320 and #1341). Timed-pregnant CD1 mice were bred and separated 12 hours later denoted as E0.5. At E9.5 or E14.5 mice were injected intravenously via the tail vein with 0.1 μg Au/g mouse of either AuNPs, NSs, or the equivalent volume of saline. Three pregnant CD-1 mice were used for each treatment group for each gestation, totaling 18 mice for this study. Twenty-four hours post-injection, mice were humanely euthanized. This timepoint was selected for analysis because 24 hours of development results in the transition to a new Theiler Stage so it would be undesirable to examine distribution and effects on fetal growth at circulation times longer than this; additionally, most AuNPs or NSs should have reached their ultimate fate by twenty-four hours post injection. Maternal blood and nonreproductive organs (liver, kidney, spleen, heart, and lung) and embryos and placentas were excised for each mouse and used for either histology or ICP-MS (discussed below). For reproductive organ analyses, two embryos (one proximal and distal to the maternal head) and their respective placentas were excised from each mouse, totaling 36 embryos and 36 placentas. Excised organs were weighed then lyophilized overnight to remove water content. Tissues were digested in 1 ml of 3% hydrochloric acid in nitric acid and heated to 60°C for 30 minutes or until completely dissolved. The digested tissue samples and standards of known gold concentration (prepared using TraceCERT® gold standard) were then diluted in a matrix containing 2% nitric acid and 2% hydrochloric acid. The gold content in each tissue sample and standard curve sample was analyzed on an Agilent 7500c ICP-MS instrument. Whole embryos and placentas and sections of maternal liver, spleen, kidneys, and lungs were excised from the pregnant mice at E10.5 or E15.5 and were fixed in 4% paraformaldehyde for 72 hours and washed in 70% ethanol twice prior to overnight processing. Processed paraffin samples were embedded and sectioned into 5-micron slices. Silver staining, which amplifies the size of NPs via deposition of silver on gold, was performed on embryos, placentas, livers and spleens to enable NP visualization by optical microscopy. Briefly, the tissue sections were deparaffinized in xylene (10 min) and hydrated through a series of ethanol (100%, 90%, 70%) and water. Silver stain (Cytodiagnostics Silver Enhancer Kit) was incubated on the slides at room temperature for 10 minutes. The samples were then rinsed and counterstained with Hematoxylin and Eosin. Finally, samples were dehydrated and mounted with xylene based mounting media and imaged using a Zeiss Axioobserver microscope. Maternal kidneys and lung sections were only stained with Hematoxylin and Eosin to examine general morphology.

AuNPs (15 nm diameter) and NSs (150 nm diameter) passivated with 5 kDa mPEG-SH as described in the Methods were characterized for their hydrodynamic diameter, zeta potential, and morphology using UV-visible spectrophotometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Spectrophotometry revealed that the AuNPs had a peak plasmon resonance at ~520 nm (not shown), consistent with the extinction properties of 15 nm diameter AuNPs [ 32 ]. Similarly, the NSs had peak plasmon resonance at ~800 nm (not shown), consistent with the extinction features of NSs with 120 nm diameter silica cores and 15 nm thick gold shells [ 33 ]. DLS measurements indicated that uncoated AuNPs had a hydrodynamic diameter of 23 ± 8 nm and a zeta potential of −23 ± 5 mV ( Figure 1A , B ). Following PEGylation, the AuNPs’ hydrodynamic diameter increased to 40 ± 7 nm and their zeta potential increased to −4.5 ± 3 mV. Since hydrodynamic diameters measured by DLS are typically larger than physical nanoparticle dimensions, the PEGylated AuNPs were further examined by TEM, which indicated the mean particle diameter was 14 ± 1 nm ( Figure 1C ). NSs were characterized similarly and found to have a hydrodynamic diameter of 150 ± 2 nm before PEG addition and 176 ± 4 nm after PEG addition ( Figure 1D ). The NSs’ zeta potential was –40 ± 0.4 mV and –3 ± 2 mV before and after PEGylation, respectively ( Figure 1E ), and TEM measurements showed the PEG-coated NSs had a mean diameter of 151 ± 13 nm ( Figure 1F ). After characterizing the NPs, we sought to examine the gestational differences in their distribution to placentas and embryos following intravenous delivery. We injected 15 nm PEG-coated AuNPs, 150 nm PEG-coated NSs, or saline at doses of 0.1 μg Au/g mouse into pregnant CD1 mice at E9.5 or E14.5 via the tail vein. Twenty-four hours later, the reproductive and major non-reproductive tissues of the mothers were excised and processed for either histological examination or quantification of gold content by ICP-MS. The ICP-MS data showed that both 15 nm AuNPs and 150 nm NSs coated in PEG accumulate to a greater extent in placentas than embryos at both E9.5 and E14.5 ( Figure 2A , B ). There is a notable decrease in the number of nanoparticles in the tissues at E14.5 relative to E9.5, indicating that particle distribution to reproductive organs is gestation-dependent ( Figure 2A , B ). The gold content in the placentas and embryos was further evaluated by histological examination of silver-stained tissue sections to corroborate the ICP-MS results. Silver stain nucleates on the surface of gold-based NPs to enable their visualization by light microscopy. Both 15 nm AuNPs and 150 nm NSs were observed in E9.5 placentas, but fewer AuNPs and NSs were observed in placentas at E14.5 ( Figure 3A , Supplemental Figure 1A , particle location indicated by white arrows ). These data confirm the quantitative ICP-MS results ( Figure 2 ) that indicated NP accumulation in placentas is gestation dependent. Within embryos, neither AuNP nor NS presence was distinct in the silver-stained tissue sections at either gestation ( Figure 3B , Supplemental Figure 1B ), in agreement with the ICP-MS results that showed both types of particles accumulate to a lesser degree in embryos than placentas. The increased sensitivity of ICP-MS relative to silver staining likely explains why we could identify particles in the embryos by ICP-MS but not by silver staining. We also investigated if AuNP or NS accumulation in embryos is a function of embryo location in the uterus. Murine ovarian and uterine arteries supply blood and nutrients to placentas, and therefore, embryos at different positions in the uterus ( Supplemental Figure 2A ). To investigate whether spatial differences exist in NP accumulation in embryos, we excised embryos and their respective placentas from the proximal (closest to ovary) or distal (closest to cervix) ends of the uterine horn and analyzed gold content in these tissues separately by ICP-MS. These two embryo positions were chosen as they represent both extremes of blood supply to the uterus; the ovarian artery supplies blood to the proximal embryos first, while the uterine artery supplies blood to the distal arteries first [ 36 ]. There was no significant difference in gold content between proximal and distal placentas or embryos at either gestational age for either NP type ( Supplemental Figure 2B , C ). This result is different than originally expected as a previous study in which researchers clamped the uterine or ovarian arteries in mice to reduce perfusion to the gravid uterus showed that the uterine arteries appeared to be more important for survival of embryos than the ovarian arteries [ 37 ]. Therefore, we expected distal embryos and placentas would have more gold content than proximal embryos and placentas. However, multiple studies have shown that the middle embryos are supplied with the least oxygen rich blood [ 34 , 35 , 38 ] compared to the distal and proximal embryos. Accordingly, while the distal and proximal embryos and placentas may have the same gold content due to their location at the blood supply entry points, as we have shown, the middle embryos may have less gold content. Future work should sample the proximal, distal, and middle embryos and placentas to test this hypothesis. To determine whether the intravenously administered AuNPs or NSs impacted fetal growth in pregnant mice, we compared the weight of embryos and placentas that were excised from pregnant mice 24 hours post-NP administration to those obtained from pregnant mice treated with saline. A 24-hour time point was chosen to ensure embryo development was not altered as 24 hours of development results in the transition into a new Theiler Stage. The embryo to placenta weight ratio (E:P), a measurement of placental efficiency, was calculated for each treatment group. An E:P ratio greater than 1, especially at late gestation, indicates efficient placental function leading to adequate fetal growth [ 38 , 39 ]. Embryos from dams treated with 15 nm PEG-coated AuNPs did not display any significant changes in E:P or mean weight when compared to saline at either gestational age ( Figure 4A , B ). Likewise, 150 nm PEG-coated NSs did not alter E:P or embryo weight at E9.5 compared to dams treated with saline, and there was no difference in E:P at E14.5 for dams treated with NSs or saline ( Figure 4C , D ). At E14.5, the embryo weights from dams treated with NSs were significantly higher than those treated with saline ( Figure 4D ). We attribute this difference to physiologic variability as the E:P ratio for NSs at E14.5 is greater than 1, indicating adequate fetal growth, and is not significantly different from that of the saline control. In aggregate, these results indicate that neither PEG-coated AuNPs nor NSs disrupt fetal growth within 24 hours of administration to pregnant mice. Given our finding that AuNP and NS accumulation in placentas and embryos is gestation dependent, we wanted to examine if their accumulation in maternal non-reproductive organs is also gestation dependent. Therefore, we excised maternal organs (lungs, liver, kidney, spleen, heart, and blood) 24 hours post-intravenous delivery of the AuNPs or NSs for analysis of gold content by ICP-MS. Analysis revealed that 15 nm AuNPs exhibit the highest accumulation in liver and spleen of pregnant mice, with no significant difference in accumulation observed for any organ tested between E9.5 and E14.5 ( Figure 5A ). Tissues excised from the dams treated with 150 nm NSs displayed similar findings, with most NSs found in the liver and spleen, and no differences in accumulation identified as a function of gestation ( Figure 5B ). These data indicate that, independent of NP size or maternal gestation, most AuNPs and NSs are filtered by the liver and spleen. The liver functions to metabolize material in blood so it is not surprising that a large portion of the NPs accumulate there. Silver staining of histological sections of the liver and spleen corroborated the ICP-MS results. Both 15 nm AuNPs and 150 nm NSs were observed in the maternal liver and spleen at both gestations while tissue morphology appeared similar to that of the saline-treated mice ( Figure 5C , Supplemental Figure 3 , representative NPs indicated by white arrows ). It should be noted that maternal blood volume increases at later gestations, so although the density of AuNPs or NSs in blood is similar between E9.5 and E14.5 mice, the total amount of NPs in blood is likely greater at E14.5; future work should confirm this experimentally by measuring total blood volume and gold content at each gestation. Our data show that PEG-coated AuNPs and NSs intravenously administered at E9.5 and E14.5 accumulate primarily in the liver and spleen at both gestational ages. These results agree with other studies that have assessed NP distribution to major non-reproductive organs at different stages of pregnancy (E5.5–13.5). For example, one study found that there were no differences in the distribution of gold NPs ranging from 1.5 nm to 70 nm in diameter to major organs (liver, spleen, kidney, lung and heart) in pregnant (E9.5) and non-pregnant mice [ 24 ]. In this study, the lung was found to be a site of high accumulation, while specific biodistribution data for the spleen was not included [ 24 ]. This data, coupled with our results, indicates that NP accumulation in non-reproductive organs is not gestation dependent.

The results of this study show that intravenously administered AuNPs and NSs can minimally accumulate in the placenta and embryo during murine pregnancy, with greater amounts of NPs distributing to the maternal liver and spleen. At both E9.5 and E14.5, more AuNPs and NSs were found in the placenta than in the embryo, and the levels of gold in these tissues decreased at E14.5 compared to E9.5, indicating that NP distribution to placentas and embryos is gestation dependent. The apparent decrease in the number of NPs/g of wet tissue at E14.5 compared to E9.5 may be attributed in part to increases in placenta and embryo weight at later gestations, as well as to altered transport mechanisms as discussed below. Importantly, the presence of the AuNPs or NSs in the body did not impact fetal development based on analysis of placental weight and embryo weight. This suggests that intravenously administered nanomedicines may be a viable treatment option for pregnancy complications in the future. The finding that both 15 nm AuNPs and 150 nm NSs coated with PEG accumulate in the placenta and fetus to a greater extent at E9.5 than at E14.5 aligns with prior work that evaluated the distribution of 13 nm AuNPs coated with PEG five hours post-intravenous delivery to pregnant mice at different gestations (E5.5-E15.5) [ 40 ]. This study showed that PEG-coated AuNPs accumulated in fetal tissues, but fetal gold concentration decreased significantly after E11.5 [ 40 ]. Our study corroborates this result and shows that a similar trend exists for larger PEG-coated NSs. We suspect dynamic changes in transplacental transport mechanisms are responsible for gestational differences in NP accumulation in placentas and fetuses. Transplacental transport can occur by active and passive mechanisms, with NPs trafficking through or between the cells that comprise the barrier. In vitro transport studies have shown that NPs may be taken up by placental synctiotrophoblasts through phagocytosis, clatherin-mediated endocytosis, cavaeolae-mediated endocytosis, or macropinocytosis [ 41 – 45 ]. After uptake, NPs typically exploit vesicular transport to transcytose across trophoblasts, although some cationic NPs may fuse directly with the basal membrane to escape the cell [ 41 , 46 ]. After crossing the trophoblast layer, NPs must diffuse through the villous stroma and pass through endothelial cell walls (again, by passive or active mechanisms) to enter fetal circulation [ 46 ]. We hypothesize that these transport mechanisms may be differentially regulated throughout gestation. Indeed, several cell membrane nutrient transporters such as cholesterol transporter SR-B1 [ 47 ] and glucose transporter GLUT4 [ 48 ] have been noted previously to be differentially expressed over gestation. Therefore, it is reasonable to surmise that endocytosis mechanisms change with advancing gestation as well. In addition, placental architecture and development dramatically change over gestation and as such, the trophoblast tissue layer may have increased permeability between cells at earlier rather than at later stages. Overall, transplacental transport and the mechanisms involved with that process are complex, metabolite/item specific, and not well understood over different phases of placental development. Further study into the mechanism behind AuNP and NS gestational dependent transplacental transport properties are needed. To bypass high amounts of hepatic clearance, local delivery routes (i.e., vaginal administration) should be investigated in the future to increase NP accumulation in reproductive organs. These studies should also be performed at various gestational ages since the properties of cervicovaginal mucus, a major barrier to vaginal drug delivery, are known to change throughout pregnancy [ 49 – 52 ]. Safety studies should also be completed to ensure NP delivery does not induce toxicity; analyses could include histological evaluation, examination of fetal development, and assessment of pup cognitive function and behavior. Future work should also investigate NP material and surface coatings as factors that can affect delivery, as the results presented here only apply to gold-based NPs coated with PEG with diameters of 15 nm or 150 nm. Finally, mechanistic transplacental transport studies are needed to better understand how NPs cross the placenta. This information would allow researchers to better engineer NPs with reduced transplacental transport and increased fetal safety. Overall, the work presented here lays the foundation for additional studies of NP-based drug delivery for the advancement of maternal and fetal health during pregnancy.

Pregnancy complications pose several challenges to the mother and developing fetus as treatments are limited due to many safety and ethical concerns. Preeclampsia [ 1 – 3 ], fetal growth restriction (FGR) [ 4 – 6 ] and placenta accreta [ 7 , 8 ] are examples of pregnancy-induced conditions that have limited treatments. As pregnancy progresses, symptoms of these diseases worsen leading to detrimental effects for maternal and fetal health. As a result, mothers often need to undergo emergency delivery via cesarean section and the newborns face additional morbidities related to their resultant premature birth. These pregnancy-related conditions warrant a dire need for therapeutic advances that can treat conditions that negatively affect maternal and fetal health during pregnancy without harm to the developing baby. Over the last decade, various nanoparticle (NP)-based carrier systems have been engineered to target a variety of diseases and cancers. It is well established that the ultimate fate of nanocarriers in the body following systemic delivery varies as a function of size, shape, and surface chemistry [ 9 , 10 ]. While researchers have harnessed this knowledge to create powerful systems for the treatment of diseases such as cervical cancer [ 11 , 12 ], endometriosis [ 13 , 14 ], and HIV [ 15 , 16 ], few have explored the potential of nanomedicine to treat pregnancy complications [ 10 , 17 ]. The state of pregnancy introduces additional factors that will influence NP distribution and hence, NP design [ 10 , 17 ]. These factors include the dynamic physiology of the maternal reproductive system [ 4 , 18 – 20 ], the transport of nutrients and drugs through the placenta [ 21 , 22 ], and the development of the fetus [ 5 , 23 ]. Understanding how these variables impact NP distribution is necessary to develop effective treatments that can extend pregnancies to term and advance maternal and fetal health. To date, many studies examining the distribution of NPs in pregnant mice or rats have utilized gold-based NPs as model carriers due to the ease of controlling their size and surface chemistry, as well as the ability to quantify gold content in tissues with high sensitivity using inductively coupled plasma mass spectrometry (ICP-MS). These studies have indicated that gold NP accumulation in reproductive tissues is size-dependent. For example, when gold nanoparticles (AuNPs) of varying sizes (1.5 nm – 70 nm) were intravenously delivered to pregnant mice, it was determined that only particles less than 10 nm could readily cross the placental barrier and enter the fetal circulation [ 24 ]. Another study evaluated AuNPs of 1.4 nm, 18 nm, and 80 nm diameter in pregnant rats and observed 1.4 nm and 18 nm AuNPs in embryos despite all sizes of AuNPs being present in pooled tissues samples consisting of the placenta, umbilical cord, and amniotic membranes [ 25 ]. Together, these findings demonstrate that size plays a critical role in NPs’ ability to reach placentas and fetuses following systemic administration [ 24 – 27 ]. Given that maternal and fetal physiology undergo dynamic changes throughout pregnancy, we wanted to determine whether the biodistribution of gold NPs in pregnant mice is gestation dependent. To investigate this question, we intravenously administered either 15 nm diameter spherical gold NPs (15 nm AuNPs) or 150 nm diameter silica core/gold shell nanoshells (150 nm NSs) to pregnant mice at gestational day (E) 9.5 or 14.5. These gestational ages were selected to represent approximately the second and third trimesters of human pregnancies based on placental development [ 28 ]. We did not choose to explore distribution at earlier timepoints because many pregnancy complications are not detected prior to the end of the first trimester, and consequently, nanomedicine-based treatment strategies would likely not be administered until the second or third trimesters. Twenty-four hours after the AuNPs or NSs were delivered via tail vein injections, mice were humanely euthanized and the maternal reproductive and non-reproductive tissues collected for both histological analysis and quantitative measurement of gold content by ICP-MS. These assays revealed that the accumulation of both 15 nm AuNPs and 150 nm NSs in placentas and embryos is gestation dependent, with more NPs found in these tissues at E9.5 than E14.5. We also found that both 15 nm AuNPs and 150 nm NSs can accumulate in these reproductive tissues without altering fetal or placental weight, and they also do not impact the histological appearance of maternal non-reproductive organs. These findings indicate that 15 nm AuNPs and 150 nm NSs have minimal short-term fetal or maternal toxicity, although the safety of these and other nanocarriers should be fully vetted in future studies that include relevant therapeutic cargo in the formulation. Overall, the results obtained from this fundamental study will guide the development of new nanomedicines to improve the treatment of pregnancy complications.