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. Author manuscript; available in PMC: 2024 Dec 1.
Published in final edited form as: Drug Deliv Transl Res. 2023 Jun 26;13(12):3003–3013. doi: 10.1007/s13346-023-01369-w

Delivery and short-term maternal and fetal safety of vaginally administered PEG-PLGA nanoparticles

N’Dea S Irvin-Choy 1, Katherine M Nelson 2, Jason P Gleghorn 1,3, Emily S Day 1,4,5
PMCID: PMC10913101  NIHMSID: NIHMS1967153  PMID: 37365402

Abstract

At the onset of pregnancy, people with preexisting conditions face additional challenges in carrying their pregnancy to term, as the safety of the developing fetus and pregnant person is a significant factor of concern. Nanoparticle (NP)-based therapies have displayed success against various conditions and diseases in non-pregnant patients, but the use of NPs in maternal–fetal health applications needs to be better established. Local vaginal delivery of NPs is a promising administration route with the potential to yield high cargo retention in the vagina and improved therapeutic efficacy compared to systemic administration that results in rapid NP clearance by the hepatic first-pass effect. In this study, we investigated the biodistribution and short-term toxicity of poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) NPs in pregnant mice following vaginal delivery. The NPs were either loaded with DiD fluorophores for tracking cargo distribution (termed DiD-PEG-PLGA NPs) or included Cy5-tagged PLGA in the formulation for tracking polymer distribution (termed Cy5-PEG-PLGA NPs). DiD-PEG-PLGA NPs were administered at gestational day (E)14.5 or 17.5, and cargo biodistribution was analyzed 24 h later by fluorescence imaging of whole excised tissues and histological sections. No gestational differences in DiD distribution were observed, so Cy5-PEG-PLGA NPs were administered at only E17.5 to evaluate polymer distribution in the reproductive organs of pregnant mice. Cy5-PEG-PLGA NPs distributed to the vagina, placentas, and embryos, whereas DiD cargo was only observed in the vagina. NPs did not impact maternal, fetal, or placental weight, suggesting they display no short-term effects on maternal or fetal growth. The results from this study encourage future investigation into the use of vaginally delivered NP therapies for conditions affecting the vagina during pregnancy.

Keywords: Nanomedicine, Pregnancy, Placenta, Embryo, Biodistribution, Maternal–fetal health, Vaginal

Introduction

Preexisting and newly emergent conditions affecting pregnant people are challenging to manage due to the limited availability of treatments with confirmed fetal and maternal safety. Without intervention, conditions including cervical cancer [1, 2], HIV [3, 4], endometriosis [5, 6], and bacterial vaginosis (BV) [7, 8] may exacerbate pregnancy challenges. Consequently, pregnant people with these and other high-risk conditions face the difficult decision to terminate the pregnancy or risk their and the unborn baby’s health by foregoing treatment for the duration of the pregnancy. These patients desperately need new treatment options that will not harm themselves or the developing fetus.

Nanoparticle (NP)-based carrier systems have emerged as effective tools for treating many diseases (e.g., cancers, COVID-19, wound healing) over the last decade. Prior advancements in nanomedicine have established that NP size, shape, and surface charge dictate biodistribution following systemic delivery [9–12]. Although researchers have applied this knowledge to treating cervical cancer [1, 2], HIV [3, 4], endometriosis [5, 6], and BV [8] in non-pregnant conditions, it remains to be established how NP treatment systems can be used for maternal–fetal health applications during pregnancy. However, maternal–fetal nanomedicine is quickly growing [9, 13–15], with a few recent studies investigating the biodistribution and safety of NPs in pregnant murine models following systemic delivery [16–18]. One study found that gold nanoparticles (AuNPs) less than 10 nm in diameter could enter fetal circulation post intravenous (IV) administration [16]. Another study determined that both 15 nm diameter AuNPs and 150 nm diameter gold nanoshells could distribute to the placentas following IV delivery, with less accumulation observed when the NPs were administered at later gestational ages [18]. These studies have demonstrated a lack of short-term toxicity due to the presence of the NPs, supporting the potential use of nanomedicine to treat maternal–fetal health conditions.

To develop locally applied nanomedicine therapy for conditions affecting the vagina during pregnancy, it is essential to understand the distribution and safety of NPs following vaginal administration. The aforementioned studies indicated that IV administered NPs could reach placentas and embryos in pregnant mice despite a majority of systemic NPs being filtered out of circulation by the mononuclear phagocyte system (MPS) [19, 20]. Systemic clearance by the MPS limits the number of NPs reaching the target tissue and increases the risk of off-target effects. Delivering NP treatments locally, directly to the tissue of interest, may help avoid these limitations to improve the management of vaginal disorders during pregnancy.

A significant challenge associated with vaginal delivery of therapeutics is the cervicovaginal mucus (CVM) barrier. CVM is a gel-like fluid comprised of glycoproteins that lines the vaginal epithelium. This fibrous network traps foreign antigens, viruses, and bacteria, preventing them from ascending the reproductive tract into the uterus [21, 22]. Previously, researchers have used neutralizing surface coatings, such as poly(ethylene glycol) (PEG), to minimize electrostatic interactions between NPs and CVM and thereby increase NP penetration through CVM [23–25]. Conversely, mucoadhesive coatings like chitosan have also been explored to increase NP retention in the vagina [1, 26, 27]. CVM viscosity and production increase over the course of pregnancy in response to hormonal changes, leading to decreased pore sizes in the fibrous networks [21, 22], which likely alters NP transport through the mucus.

Given the physiological changes of CVM during pregnancy, we evaluated the biodistribution of PEG-poly(lactic-co-glycolic acid) (PEG-PLGA) copolymer NPs following vaginal delivery in pregnant mice at two different gestational ages. PEG-PLGA NPs were used in this study because PLGA is a Food and Drug Administration-approved, hydrolytically degradable polymer that can encapsulate diverse types of cargo and can be easily modified with PEG to support CVM penetration. We vaginally delivered saline or PEG-PLGA NPs loaded with DiD fluorophores (DiD-PEG-PLGA NPs) to pregnant mice at gestational day (E)14.5 or E17.5 corresponding to early and late time points in the third trimester of human pregnancy, respectively. We also administered Cy5 labeled PEG-PLGA NPs (Cy5-PEG-PLGA-NPs) to separate mice at gestational age E17.5 to evaluate polymer distribution following vaginal administration. Immediately after NP or saline administration, the mice were fluorescently imaged with an in vivo imaging system (IVIS) to confirm successful injection. Twenty-four hours later, whole-body images were taken, and the major maternal and reproductive organs were excised and analyzed by IVIS and cryo-histology. These experiments revealed that DiD cargo was retained only in the vagina and did not distribute to other organs within 24 h of injection, whereas Cy5-labeled PLGA was observed in the vagina, placentas, and embryos. There were no significant differences in DiD signal in the vagina between gestational ages, indicating cargo biodistribution is not dependent on gestational age when vaginally delivered to pregnant mice. Importantly, vaginally administered NPs displayed no short-term effects on maternal and fetal growth and safety; however, future studies should more extensively investigate the long-term fetal effects of PEG-PLGA NPs and their efficacy as a treatment when loaded with therapeutic molecules. Overall, these findings indicate the potential for PEG-PLGA NPs in treating maternal conditions affecting vaginal health during pregnancy.

Methods

Synthesis of DiD-Loaded PEG-PLGA NPs

DiD-loaded PEG-PLGA NPs were synthesized by a single emulsification method. First, 50:50 PLGA (32 kDa) was dissolved in acetone at 1 mg/mL. Next, PEG (5 kDa)-PLGA (30 kDa) was dissolved in dichloromethane (DCM) at 1 mg/mL and then added at a 1:3 volumetric ratio to the PLGA/acetone solution. Fluorescent DiD was dissolved in dimethyl sulfoxide (DMSO) at 9 mM, and 0.5–3 μL of this solution was added to the PLGA/PEG-PLGA solution. Lastly, the PLGA/PEG-PLGA/DiD solution was added dropwise to 0.2% poly-vinyl alcohol (PVA) in MilliQ H2O at a 3:1 volumetric ratio. NPs were magnetically stirred for 2 h at 800 rpm to allow solvent evaporation, then centrifuge filtered with 50 kDa molecular weight cutoff (MWCO) conical filter tubes for 25 min at 4200 rpm. The DiD-PEG-PLGA NPs were resuspended in 3 mL of MilliQ H2O and centrifuged a second time for 30 min or until most of the water was removed. NPs were then diluted in phosphate-buffered saline (PBS) to a fluorescent value of at least 10,000 (measured by reading 100 μL of sample in a 96-well plate on a BioTek Synergy H1M plate reader with excitation/emission of 640 nm/670 nm) and transferred to Eppendorf tubes for a maximum of 2 h before being delivered to mice.

After synthesis, an alternative filtration method was used to remove additional DiD from NP samples. Following the initial centrifugation in 50 kDa MWCO conical filters for 25 min, NPs were resuspended in 500 μL of 0.5% Triton X-100 to disrupt any DiD micelles that may have formed during synthesis. Subsequently, the NPs were centrifuge filtered in 50 kDa MWCO conical filters for 15 min at 4200 rpm. NP washes were performed three times by resuspending NPs in 2 mL of MilliQ H2O between each centrifugal filtration step.

Synthesis of Cy5-PEG-PLGA NPs

Cy5-PEG-PLGA NPs were synthesized also using a single emulsification method. First, Cy5-PLGA and PLGA (32 kDa) were dissolved in acetone in separate scintillation vials at 1 mg/mL concentrations. PEG (5 kDa)-PLGA (30 kDa) was dissolved in DCM at 1 mg/mL and 250 μL of this solution was transferred to a clean Eppendorf tube. Next, 650 μL of the PLGA/acetone solution and 100 μL of the Cy5-PLGA/acetone solution were transferred to the Eppendorf tube already containing the PEG-PLGA/DCM solution for a total of 1 mL of solution. The Cy5-PLGA/PLGA/PEG-PLGA solution was then added dropwise to 3 mL of 0.2% PVA in MilliQ H2O while magnetically stirring at 800 rpm. NPs were covered with aluminum foil and stirred overnight. Following stirring, Cy5-PEG-PLGA NPs were centrifuge filtered in 100 kDa MWCO filters for 25 min at 4200 rpm and 14 °C. The Cy5-PEG-PLGA NPs were resuspended in 3 mL of MilliQ H2O then centrifuge filtered a second time for 30 min at 4200 rpm and 14 °C. To prepare NPs for mouse injections, the fluorescence of 100 μL of Cy5-PEG-PLGA NPs was measured using a BioTek Synergy H1M plate reader at excitation/emission wavelengths of 640/670 nm and then diluted in PBS to a fluorescent value of 10,000. Cy5-PEG-PLGA NPs were stored in dark Eppendorf tubes at 4 °C for a maximum of 2 h before use.

Nanoparticle characterization

Following synthesis, DiD-PEG-PLGA NPs and Cy5-PEG-PLGA NPs were characterized by dynamic light scattering (DLS) and zeta potential measurements on a Litesizer500 instrument (Anton Paar). To prepare NP samples, 5–10 μL of NPs were diluted in 1 mL of MilliQ H2O in disposable or Omega cuvettes (Anton Parr) for DLS or zeta potential measurements, respectively. The reported intensity-based hydrodynamic diameter and the mean zeta potential are the averages of three DLS experiments. NP diameter and morphology were also evaluated by transmission electronic microscopy (TEM). TEM grids were stained with 2% uranyl acetate, dried, and examined with a Zeiss Libra 120 Transmission Electron Microscope.

Evaluation of DiD encapsulation and release from PEG-PLGA NPs

DiD loading in PEG-PLGA NPs was determined by dissolving fully synthesized NPs in DMSO and measuring the resulting fluorescence. Purified DiD-PEG-PLGA NPs were dissolved in 500 μL of DMSO and placed on a rocker at room temperature for 30 min. Next, the solution of dissolved NPs and previously encapsulated DiD was centrifuged at 14,000 rpm for 30 min to separate the DiD from pelleted PEG-PLGA fragments. Following centrifugation, the fluorescence of the supernatant was measured in a Synergy H1 plate reader at an excitation/emission of 640/670 nm, respectively. All samples were compared to a standard curve of known DiD concentrations to calculate the amount of dye encapsulated. Encapsulation efficiency was found by dividing the measured amount of encapsulated dye by the known amount of dye added during synthesis.

To determine dye release from DiD-PEG-PLGA NPs in different conditions, the NPs were transferred to 1 mL of MilliQ H2O and stored at 4 °C (storage conditions) or to 1 ml of 137 mM NaCl at pH 3.5 while shaking at 37 °C (to represent the human vaginal environment) [28]. At 1, 2, 4, and 24 h, the NPs were centrifuged for 20 min at 14,000 rpm to pellet the NPs. The supernatant with released DiD was removed and 100 μL was added to each well in a 96-well plate. As DiD is weakly fluorescent in water, 100 μL of DMSO was added to each well to allow measurement of the molecule’s absorbance at 640 nm (which correlates with concentration) using a Synergy H1 plate reader. The NP pellet was resuspended in fresh solution, and the process was repeated at the remaining time points. After 24 h, the remaining dye encapsulated was quantified by dissolving the NPs in DMSO, as described above. DMSO was added to all NP samples and compared to a standard curve of known DiD concentrations in DMSO/solvent to calculate the dye release over 24 h.

In vivo pregnancy murine model and IVIS imaging

Female mice between 8 and 17 weeks of age 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). Virgin CD1 mice were bred and separated 12 h later, denoted as E0.5. Mice were regularly weighed throughout the study to monitor pregnancy progression. At E14.5 or E17.5, mice were injected intravaginally with 20 μL of either saline or freshly synthesized DiD-PEG-PLGA NPs at a fluorescence of at least 10,000 relative fluorescence units but less than 40,000 (measured in the plate reader as described above). Separate mice at E17.5 were injected intravaginally with 20 μL of either saline or freshly synthesized Cy5-PEG-PLGA NPs at a fluorescence of 10,000 (measured in the plate reader as described above). To achieve a successful injection, mice were anesthetized with inhaled isoflurane. Anesthetized mice were placed dorsally, and the NP or saline solution was intravaginally injected using a glass microcapillary pipette. Mice were left in the dorsal position for at least 5 min after injection to ensure the solution remained in the vaginal canal. Mice were then moved to an in vivo imaging system (IVIS, Lumina III, Perkin-Elmer) to confirm successful injection via fluorescent detection of the DiD-loaded NPs or Cy5-labeled NPs (excitation/emission 640 nm/670 nm). Twenty-four hours post-injection, mice were humanely euthanized. Whole-body IVIS images were taken, then maternal organs (liver, kidney, spleen, heart, lung, vagina, and ovaries), embryos, and placentas were excised and IVIS imaged to detect the presence of a fluorescent signal. Subsequently, the embryos and placentas were counted and weighed, and all tissues from DiD-PEG-PLGA NP injected mice were flash-frozen to prepare cryosections (discussed below). IVIS images were analyzed in Living Image (Perkin-Elmer), and region of interest (ROI) areas were drawn around vaginas to measure their fluorescence.

Qualitative evaluation of DiD cargo presence in cryosections

The major maternal organs (liver, spleen, heart, kidney, and lungs) and reproductive organs (vagina, ovaries, embryos, and placentas) excised from the saline or DiD-PEG-PLGA NP injected pregnant mice at E15.5 or E18.5 were flash-frozen in isopentane to encourage uniform tissue freezing and limit cracking. First, isopentane was transferred to a metal container and chilled for 10 min in a Styrofoam box of approximately 15 mL of liquid nitrogen. Once the isopentane was chilled, tissues were transferred into it for 30–45 s or until frozen. Immediately after freezing, organs were cryo-embedded in optimal cutting temperature (OCT) compound and temporarily stored on ice. The embedded tissues were sliced into 5 μm sections using a CryoStat Leica CM3050S at 24 °C. Tissue sections were washed in PBS once for 5 min and air-dried while covered. Samples were immediately imaged using a Zeiss Axio Observer microscope and examined for fluorescent DiD signal in the DiD channel (670 nm) and general morphology in brightfield.

Results

Nanoparticle characterization

DiD-PEG-PLGA NPs were synthesized (Fig. 1A) and characterized for their hydrodynamic diameter, zeta potential, encapsulation efficiency, and dye release profiles using DLS, TEM, and a fluorescence plate reader. The NPs were spherical in morphology per TEM analysis (Fig. 1B), with a hydrodynamic diameter of 106 ± 3 nm and zeta potential of − 19 ± 0.3 mV (Fig. 1C). When 1 μl of 9 mM DiD was added during the synthesis procedure, the encapsulation efficiency was 39 ± 6%, equating to 3.7 ± 0.5 μg of dye encapsulated per mg of PEG-PLGA (Fig. 1C). DiD saturation was also evaluated by loading different volumes (0.25, 0.5, 1, 2, 3 μl) of 9 mM DiD during NP synthesis. This analysis determined that dye volume did not affect the hydrodynamic diameter or zeta potential of the NPs (Supplemental Fig. S1). It also indicated that adding more than 1 μl of dye did not increase NP fluorescence despite higher loading, suggesting that quenching may occur above this threshold (Supplemental Fig. S1). DiD release kinetics were evaluated in storage conditions (water, 4 °C, pH 7) and conditions approximating the acidic human vaginal microenvironment (NaCl, 37 °C, pH 3.5) over 24 h using absorbance values of dye no longer encapsulated in the NPs. The absorbance measurements revealed that 90 ± 6% of the encapsulated DiD was released at 24 h under physiologic conditions, whereas 78 ± 4% of the dye was released under storage conditions (Fig. 1D). Together, these data indicate that DiD was successfully encapsulated in the PEG-PLGA NPs, and more dye was released over 24 h in physiologic conditions. This agrees with previous research that shows cargo release is accelerated from PEG-PLGA NPs placed in acidic and/or elevated temperature conditions [29–31]. Cy5-PEG-PLGA NPs were synthesized as described (Fig. 1E) and characterized for their hydrodynamic diameter and zeta potential (Fig. 1F). Compared to DiD-PEG-PLGA NPs, the Cy5-PEG-PLGA NPs had a slightly larger hydrodynamic diameter of 139 ± 25 nm and a similar zeta potential of − 15 ± 2 mV across four batches (Fig. 1F). The hydrodynamic diameter and zeta potential of the specific batch of NPs injected into the mice was 165 ± 20 nm and − 20 ± 2 mV.

Fig. 1.

Fig. 1

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Synthesis and characterization of DiD-PEG-PLGA NPs and Cy5-PEG-PLGA NPs. A Schematic of DiD-PEG-PLGA NP synthesis. B Transmission electron micrograph of NPs. C Hydrodynamic diameter, zeta potential, encapsulation efficiency, and DiD loading of NPs after synthesis and removal of excess dye by suspension in Triton X-100 and centrifugal filtration. Data indicate mean ± standard deviation. n = 4 D DiD release profile over 24 h under storage (pH 7.0, 4 °C) and physiologic (pH 3.5, 37 °C) conditions. Data are mean ± standard deviation of n = 3 experiments. E Schematic of Cy5-PEG-PLGA NP synthesis. F Hydrodynamic diameter and zeta potential of Cy5-PEG-PLGA NPs following synthesis and purification. Data show mean ± standard deviation of n = 4 experiments

DiD-PEG-PLGA NPs administered vaginally at E14.5 and E17.5 show localized fluorescence in the vagina 24 h post-injection

After the DiD-PEG-PLGA NPs were characterized, we examined DiD distribution in pregnant mice following vaginal delivery at gestational ages E14.5 and E17.5. Using a glass microcapillary pipette, we injected 20 μL of saline or DiD-PEG-PLGA NPs suspended in 1 × PBS into pregnant CD1 mice (Fig. 2A). Whole-body images of mice were taken by IVIS immediately following the injection and 24 h later, at gestational ages E15.5 and E18.5. IVIS imaging of the injected mice showed that fluorescence was present in the vagina immediately after delivery, confirming the DiD-PEG-PLGA NPs were injected successfully (Fig. 2B). After whole-body IVIS images were taken 24 h after injection, the mice were sacrificed, and major organs (liver, spleen, heart, kidney, lungs, vagina, ovaries) and placentas and embryos were excised, imaged, and processed for histological examination. IVIS imaging determined that DiD was present in the vagina 24 h post-injection at both gestational ages but was not seen in any other organs (Fig. 3A). Quantification of the average radiant efficiency in the vaginal tissue confirmed NP injected mice displayed a significantly higher fluorescence than the saline mice at both gestational ages (Fig. 3B). No significant difference in fluorescence between gestational ages for NP-treated mice was observed. To corroborate that the observed signal was not due solely to unencapsulated free dye remaining in the NP solution after synthesis, the experiment was repeated with NPs that were further purified by suspension in 0.5% Triton X-100 and washing several times by centrifugal filtration before vaginal injection into E14.5 mice. The signal from vaginas treated with purified NPs was similar to previous findings (Supplemental Fig. S2). However, considering the quick release of DiD from the PEG-PLGA NPs in physiological conditions (Fig. 1D), we decided to conduct further analyses with Cy5-PEG-PLGA NPs to understand where the polymer distributes to after vaginal NP administration, as well as further histological analysis to understand DiD cargo distribution within tissues, particularly the vagina.

Fig. 2.

Fig. 2

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Vaginal delivery method. A Experimental timeline, from vaginal injection at t = 0 h through tissue collection and imaging at t = 24 h. B Whole body IVIS imaging of E14.5 mice at t = 0 h after DiD-PEG-PLGA NP or saline injection

Fig. 3.

Fig. 3

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Assessment of DiD cargo accumulation in maternal organs. A IVIS imaging of maternal non-reproductive and reproductive organs taken 24 h post-DiD-PEG-PLGA NP or saline administration at E14.5 or E17.5. B Average radiant efficiency of vaginas excised from mice 24 h after DiD-PEG-PLGA NP or saline administration at E14.5 or E17.5. *indicates p < 0.05 by t test. There were no significant differences in vaginal radiant efficiency between gestational ages (i.e., between E14.5 and E17.5 mice exposed to NPs) as confirmed by a t test. n = 6 mice per group

DiD presence in the maternal organs was evaluated by histological examination of cryo-sectioned tissues to validate and expand upon the IVIS results. Tissues were flash-frozen in isopentane, cryo-embedded in OCT, cryo-sectioned, and imaged the same day to observe DiD signal. Fluorescence microscopy confirmed DiD signal was present only in the vagina and no other reproductive or major maternal organs. Specifically, DiD signal was present along the vaginal canal, nearest the vaginal opening (the site of injection) (Fig. 4). DiD fluorescence was not observed at or beyond the cervix, indicating DiD remained in the vagina over the 24-h period after vaginal administration. The livers and spleens were also evaluated for fluorescence by histology, as NPs are known to distribute to these organs following IV injections. Microscopic examination of the livers and spleens indicated DiD signal was absent in these organs at either gestational age (Supplemental Fig. S3). The ovaries were also assessed by cryo-histology since previous research has shown that IV-administered NPs distribute to the reproductive tissues in non-pregnant mice during the estrus phase of the estrus cycle [32]. In our study, we assessed all reproductive tissues at two different stages of pregnancy. We determined via histological examination that DiD did not reach the ovaries of pregnant mice within 24-h following vaginal administration at either gestational age E14.5 or E17.5 (Supplementary Fig. S4). These data confirm the quantitative IVIS data that showed DiD is present only in the vagina after vaginal NP administration.

Fig. 4.

Fig. 4

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Fluorescence microscopy of cryo-sectioned vaginas that were excised from mice 24 h post-NP or saline administration. A Tile images (× 5 magnification) of excised vaginas. Arrows indicate vaginal opening where NPs were injected. Brackets indicate region of the vaginal canal and the dotted line denotes cervix separation. *indicates full vaginal opening is not visible in image. Boxes in blue and red indicate areas shown in magnified images in B. B Magnified images (× 20) of vaginal regions indicated by blue or red boxes in A. In A and B, red indicates DiD fluorescence

To ensure DiD did not distribute to the embryos, we examined the placentas and embryos excised at E15.5 and E18.5 by IVIS imaging and histology. IVIS images of the placentas and embryos revealed that DiD signal was absent in fetal tissues (Fig. 5A). Histological examination of the placentas and embryos confirmed these findings in that the DiD signal was not observed at either gestational age (Supplemental Figure S4). Taken together, these data suggest DiD cargo distributes only to the vagina following vaginal NP delivery at E14.5 and E17.5.

Fig. 5.

Fig. 5

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Evaluation of NP distribution to placentas and embryos and NP effects on short-term embryo and placenta growth. A IVIS imaging of representative embryos and placentas excised from pregnant mice 24 h after saline or NPs were injected on E14.5 and E17.5. B Embryo-to-placenta weight ratio (E:P ratio) from mice treated with NPs or saline at each gestational age. Data show mean ± standard deviation. n = 6 litters of 307 embryos and 307 placentas total. A Student t test determined no significant differences between the NP and saline-treated mice

Cy5-PEG-PLGA NPs administered vaginally at E17.5 exhibit fluorescence in vaginas, placentas, and embryos 24 h post-injection

To determine the distribution of the polymer within the NPs following vaginal administration, we synthesized Cy5-PEG-PLGA NPs using Cy5-labeled PLGA. Unlike the DiD cargo, which is rapidly released from DiD-PEG-PLGA NPs within the 24-h observation period, the Cy5 fluorophore remains tethered to the PLGA, allowing for tracking of the Cy5-PEG-PLGA NPs over the 24 h following administration. Cy5-PEG-PLGAs were administered vaginally to pregnant CD1 mice at E17.5 and IVIS images from the NP-injected mice revealed Cy5 signal was present in the vagina (Fig. 6A). Cy5 signal was also observed in placentas and embryos (Fig. 6B). Quantitative analysis determined that the average radiant efficiencies of the vagina, placentas, and embryos were significantly higher for the NP-injected mice compared to the saline-injected mice (Fig. 6C). Collectively, these data suggest that PEG-PLGA NPs can distribute to the vagina, placenta, and embryo following vaginal administration, but that cargo that is rapidly released from these NPs in the acidic vaginal environment is strictly retained in the vagina.

Fig. 6.

Fig. 6

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Assessment of Cy5-PEG-PLGA NP accumulation in maternal organs and embryonic tissues. A IVIS imaging of maternal non-reproductive and reproductive organs taken 24 h post-NP or saline administration at E17.5. B Average radiant efficiency of vaginas, placentas, and embryos excised from mice 24 h after administration of NPs or saline at E17.5. *indicates p < 0.05, **** indicates p < 0.0001 by t test. n = 1 saline mouse with 9 placentas and embryos, n = 3 NP injected mice with a combined total of 41 placentas and embryos

DiD-PEG-PLGA NPs do not directly affect in utero fetal and maternal growth over 24 h of exposure

Gross morphological examination of the embryos and placentas ensured no major defects were seen due to NP administration. No gross differences were observed between embryos and placentas from NP-treated dams compared to saline-treated dams at either time point (Fig. 5A). To determine if the vaginal administration of DiD-PEG-PLGA NPs to pregnant mice affected fetal or maternal growth, we calculated the embryo-to-placenta (E:P) weight ratio of saline and NP-injected mice. The E:P weight ratio is a measure used to determine the overall placental efficiency in utero. An E:P ratio greater than one indicates an efficient placenta and adequate fetal growth [33, 34]. Our study showed no significant differences between the E:P ratios of the mice treated with saline and those treated with DiD-PEG-PLGA NPs for either gestational age (Fig. 5B). The maternal weight gain was also evaluated over the pregnancy and injection period to observe potential maternal stress. The DiD-PEG-PLGA NP treatment group did not display any significant differences in overall maternal weight gain throughout gestation compared to the saline treatment group in either gestational age (Fig. 7A). Additionally, there were no significant differences between the saline and NP groups in maternal weight gain within the 24-h treatment period at either gestational age (Fig. 7B). The number of resorptions per litter was also quantified during dissection to ensure DiD-PEG-PLGA NPs did not cause acute fetal distress. Here, it was determined that the number of resorptions in the NP treatment group was not significantly different from that of the saline treatment group (Fig. 7C). Similarly, there were no significant differences in the number of embryos in each litter between the treatment groups (Fig. 7D). These results en masse indicate that DiD-PEG-PLGA NPs do not cause any short-term, direct effects on maternal or fetal growth.

Fig. 7.

Fig. 7

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Short-term effects of NPs on maternal and fetal growth. A Maternal weight gain from embryonic day 0 to the end of the study. B Maternal weight change over the 24 h after NP or saline injection. C The number of embryonic resorptions per litter in each treatment group. D Number of embryos in utero per liter. There were no significant differences between the treatment groups across any of these metrics, as confirmed by a t test comparing saline to NP-exposed mice. Data show mean ± standard deviation. n = 6 pregnant mice per group, with a total of 307 embryos

Discussion

This study shows that DiD released from vaginally administered PEG-PLGA NPs is retained in the vagina and does not accumulate in other organs when administered at E14.5 and E17.5 gestational ages of murine pregnancy (Figs. 3 and 4). Quantitative IVIS analysis indicated no significant difference in DiD signal in the vaginas between E14.5 and E17.5 NP-treated mice (Fig. 3), indicating DiD cargo retention in the vagina is not gestation dependent at these time points. Interestingly, the quantitative analyses of Cy5-PEG-PLGA NP distribution 24 h after vaginal administration at E17.5 revealed that the NPs could accumulate in the vagina, placenta, and embryo (Fig. 6). There was no effect of the DiD-PEG-PLGA NPs on maternal weight gain indicating they did not influence maternal wellbeing over the duration of the study compared to the saline-injected dams (Fig. 7). Further, the NPs did not impact fetal development based on the placenta and embryo weights and morphology (Fig. 5). Lastly, dams injected with NPs did not display differences in litter size or resorption number compared to saline-injected dams (Fig. 7). These data suggest the potential to use PEG-PLGA NPs as vaginal delivery vehicles to treat conditions affecting the vagina during pregnancy.

We observed that DiD encapsulated in PEG-PLGA NPs is retained in the vagina for 24 h after vaginal administration, consistent with other studies that have evaluated polymer-based NP retention following vaginal administration in non-pregnant mice [23]. Cu et al. delivered coumarin-loaded PEG-PLGA NPs vaginally to mice during the diestrus phase to correspond to the follicular phase or the onset of menstruation in humans for the vaginal delivery of therapeutics. Our study shows similar results to the Cu study but was performed in pregnant mice at E14.5 and E17.5 gestational ages and adds new information about polymer distribution (based on Cy5-PLGA) compared to cargo distribution (based on DiD). Notably, cargo retention in the vagina is likely due to entrapment within the cervicovaginal mucus. Numerous studies [25, 35] have shown that NP physicoochemical properties, such as size, surface charge, PEG coating density, and molecular weight, influence transport within CVM. Therefore, future studies that build on our work could seek to understand how different PEG coating densities, alternative surface coatings, and larger and smaller diameters affect NP retention time in the vagina and distribution to placentas and embryos during pregnancy. Also, to ensure local delivery is feasible clinically, the length of time that the particles and their cargo are retained in the vaginal canal should be evaluated in vivo and in clinical settings; our study only investigated 24 h post-delivery because the transition to a new Theiler Stage occurs after 24 h of development. Characterization of the stability of the particles in vivo over extended periods of time would be informative for drug formulation.

Previous research has shown that NP distribution in the pregnant murine model is gestation-dependent following IV delivery [16–18]. Given these findings and the physiological changes in CVM throughout pregnancy, we expected DiD cargo retention following vaginal NP delivery would also be gestation-dependent. While E14.5 and E17.5 both represent the third trimester of human pregnancy, murine CVM continues to thicken as pregnancy progresses [36]. We hypothesized that DiD retention would differ as gestational age increased. However, quantifying IVIS images determined no difference in DiD signal in the vaginas between the E14.5 and E17.5 NP mice. Additionally, the histological examination showed no notable differences in vaginal DiD signal between the two gestational ages. These data indicate that gestational age does not play a role in DiD retention in the vagina after NP delivery in the pregnant murine model. This may be due to the rapid release of the DiD from the NPs or to minimal changes in CVM properties between E14.5 and E17.5. Future work could characterize murine CVM pore size, viscosity, and other characteristics as a functional of gestational age to shed light on the results observed here.

Our study was focused on examining significant metrics of short-term toxicity. In the future, the long-term effects of local NP administration should be evaluated to ensure fetal and maternal safety. The analyses should include an evaluation of liver enzymes to indicate liver toxicity, an assessment of serum cytokines in pregnant subjects to reveal any immunogenicity, and an analysis of fetal development, fetal immune response, and neonatal cognitive function and behavior. To explore the potential of vaginally administered NP treatments, PEG-PLGA NPs should be loaded with a therapeutic agent, and NP efficacy and stability should be evaluated in vitro and in vivo. The knowledge acquired would allow researchers to develop nanocarriers that can effectively treat maternal conditions during pregnancy while maintaining the safety of the developing fetus and pregnant people. In conclusion, the work shown here is foundational for the future development and use of nanomedicine in maternal–fetal applications.

Supplementary Material

Supporting Information

Acknowledgements

The authors thank Shannon Modla for assistance acquiring TEM images and Maneesha Sahni for assistance with Cy5-PEG-PLGA NP synthesis.

Funding

This work was supported by the National Institutes of Health under grant numbers R35GM119659 (ESD), U54GM104941 (ESD, JPG), U19AI158930 (JPG) and T32GM133395 and F31HD105398 (KMN). NIC received support from the University Graduate Scholars Program. TEM access was supported by grants from the NIH-NIGMS P20 GM103446, the NSF (IIA-1301765) and the State of Delaware. The content of this article is solely the responsibility of the authors and does not necessarily reflect the views of the funding agencies.

Footnotes

Competing interests The authors declare that they have no competing interests.

Animal studies All institutional and national guidelines for the care and use of laboratory animals were followed.

Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s13346-023-01369-w.

Availability of data and materials

The datasets generated during the current study are available from the corresponding authors on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
NIHMS1967153-supplement-Supporting_Information.docx (1.2MB, docx)

Data Availability Statement

The datasets generated during the current study are available from the corresponding authors on reasonable request.

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