Objective
The objective of the study was to examine whether the size of silicon nanovectors (SNVs) inhibits their entrance into the fetal circulation.
Study Design
Pregnant rats were intravenously administered with SNVs or saline. The SNVs were spherical particles with 3 escalating diameters: 519 nm, 834 nm, and 1000 nm. The maternal and fetal distribution of SNVs was assessed.
Results
In animals that received 1000 or 834 nm SNV, silicon (Si) levels were significantly higher in the maternal organs vs the saline group, whereas the silicon levels in fetal tissues were similar to controls. However, in animals receiving 519 nm SNVs, fetal silicon levels were significantly higher in the SNV group compared with the saline group (5.93 ± 0.67 μg Si per organ vs 4.80 ± 0.33, P = .01).
Conclusion
Larger SNVs do not cross the placenta to the fetus and, remaining within the maternal circulation, can serve as carriers for harmful medications in order to prevent fetal exposure.
A longstanding problem in the field of obstetrics has been the limitations of medication and drug use in pregnant women. Medications are frequently used during pregnancy to treat maternal medical conditions or pregnancy complications. Up to 96.9% women report consuming at least 1 medication during their pregnancy. Multiple drug use occurs in 33.5% of women, with up to 13.6% consuming 4 or more medications. Although the safety of many medications has been proven in pregnancy, there are some that cause birth defects and are therefore contraindicated in pregnancy. Examples include warfarin, certain antiepileptic medications, and isotretinoin.
Fetal exposure to potentially harmful medications is based on the ability of medications to cross the placenta from mother to fetus. The most common form of passage of medications across the placenta is by passive or simple diffusion. By this method, the amount of medications that does cross the placenta is dependent on the concentration of the medication in the maternal circulation. Although there are other important factors including physical and chemical properties of the drug, size is a strong predictor of successful transfer across the placenta. Medications that are less than 500 Da have varying levels of transfer, whereas those greater than 6000 Da such as low-molecular-weight heparin rarely pass.
Nanotechnology deals with materials and systems whose structure and components exhibit novel and significantly improved physical, chemical and biological properties, phenomena, and processes because their size ranges between 10 −9 to 10 −7 meters. Nanovectors, serving as the missing size link between the biological objects of micron scale (eg, living cells) and the therapeutic molecules (10 –10 meter scale), have emerging biological behaviors that may be utilized to enhance the therapeutic potential and to prevent side effects of currently used drugs.
As an example, nanovectors have the potential to target drug delivery to the specific location in the body, based on their physicochemical and geometrical features or specific ligands to the receptors overexpressed on the target tissue. Porous silicon-based nanovectors are widely studied for use in medicine because they can degrade in the body to naturally occurring silicic acid and can be readily modified to target specific organs. From an obstetrical perspective, such nanodevices can be developed to prohibit the transfer of medications from mother to fetus. If these nanoparticles can indeed remain within the maternal circulation, then teratogenic medications that were previously contraindicated in pregnancy could be used. This could lead to new opportunities in the medical treatment for pregnant women.
The objective of this study was to determine whether the diameter of silicon nanovectors (SNVs) may restrict their distribution to only the maternal circulation and thereby inhibit their entrance into the fetal circulation.
Materials and Methods
Timed pregnant Sprague Dawley rats were obtained from Harlan Sprague Dawley (Indianapolis, IN) on gestational day (GD) 15. Rats were individually housed in polycarbonate cages in an environmentally controlled vivarium under 12 hour light and dark cycles. Animals were fed ad libitum throughout the experiment. The animal care and experiments were approved by the University of Texas Health Science Center at Houston Animal Welfare Committee.
On GD 20, animals were administered either SNVs dispersed in 1 mL saline or saline only via jugular vein injection. Three different sizes of spherical SNVs tagged with fluorescein were tested: 519 nm, 834 nm, or 1000 nm in diameter. For characterization purposes, the particles were visualized under FEI Quanta 400 field emission-scanning electron microscope (FEI Co, Hillsboro, OR), and the surface zeta potential of the particles was analyzed using a Zetasizer nano ZS (Malvern Instruments Ltd, Southborough, MA). The particles as visualized under the scanning electron microscope and their surface charges are shown in Figure 1 .
The concentration injected was 1.2 × 10 –9 in each pregnant rat in 1 mL volume via jugular vein injection. Four hours after SNV administration, laparotomy was performed under isofluorane anesthesia and the maternal livers, uteri, placentas, and fetuses were retrieved and weighed. Then the maternal liver, uteri, and placentas were randomly divided into 2 parts: 1 for elemental analysis of silicon (Si) and 1 for histological evaluation and identification of fluorescent SNV distribution in the tissue following cryosectioning. For the fetus, 2 random whole fetuses were used for elemental analysis of the silicon, histologic evaluation, and fluorescent SNV distribution each.
Silicon levels were quantitatively measured in each organ using inductively coupled plasma mass spectrometry. The parts of the organs intended for analysis of Si were weighed, digested in Proteinase K solution, and left for up to 48 hours at 55°C for extraction of Si. Then the extracts were centrifuged at 4200 rpm for 25 minutes, and 0.5 mL of the supernatant were withdrawn, diluted with 2.5 mL of deionized water, and analyzed for Si contents.
Silicon contents were measured using a Varian Vista-Pro inductively coupled plasma atomic emission spectrometer housed in the Rice University Geochemistry Laboratory. Silicon was detected at the following wavelengths: 250.69, 251.43, 251.61, and 288.158 nm. Six standards were prepared using 1 ppm sodium silicate as a stock solution and 18 μΩ water as a diluent. Yttrium (1 ppm) was added to both standards and samples to correct for instrumental drift during the run. A calibration run including the internal control was made before each group of 15 samples.
In addition, samples were analyzed in random order to avoid any bias in data acquisition. The detection limit of Si was 15 ppb. For measurement of the total silicon in each sample (100%), the original particles suspensions were diluted and dissolved in 1 N NaOH for 24 hours at 37°C. Furthermore, all results were recalculated considering the dilutions performed.
Tissue distribution of SNVs was qualitatively assessed using fluorescence microscopy identifying the absence or presence of SNVs (tagged with fluorescein [FITC]) within each organ. Tissue was stained with 4′,6-diamidino-2-phenylindole (DAPI) fluorescent stain to identify nuclear structures of cells. Images were taken with the BX51 fluorescent microscope (Olympus, Tokyo, Japan) using filters for DAPI and FITC at ×100 magnification.
Analysis of variance was used to compare the differences in silicon concentrations in each tissue based on the 3 different SNV sizes. The Student t test was used to compare differences in silicon concentrations between individual SNV sizes and control. Fetal silicon levels were measured in 2 random fetuses per litter obtained from a single pregnant rat within each SNV group. Silicon concentrations were calculated as micrograms of silicon per gram of tissue and are presented as mean ± SD. A P < .05 was considered significant.
Results
Six animals received 1000 nm SNVs, 6 received 834 nm SNVs, 6 received 519 nm SNVs, and 5 received saline. The mean weights for each organ are as follows: maternal liver, 14.2 ± 7.5 g; maternal uterus, 6.0 ± 2.6 g; placenta, 0.9 ± 0.5 g; and fetus, 1.7 ± 0.9 g. The number of pups in each group is as follows: 1000 nm SNV, 11.7 ± 3.7 pups; 834 nm SNVs, 13.7 ± 1.6 pups; 519 nm SNVs, 14.5 ± 2.3 pups; and the saline group, 13.2 ± 1.3 pups. There were no maternal deaths or stillbirths.
Silicon levels in maternal organs and the fetus are summarized in the Table . In animals that received 1000 nm and 834 nm SNVs, silicon levels were significantly higher in the livers and uteri of the SNV group vs saline ( Figure 2 ). In contrast, placental and fetal silicon levels were similar in both groups. This demonstrates that in these large-size groups, the SNVs did not cross the placenta to the fetus but remained within the maternal circulation.
| Variable | 1000 nm (n = 6) | 834 nm (n = 6) | 519 nm (n = 6) | Control (n = 5) | P value |
|---|---|---|---|---|---|
| Maternal liver | 32.67 ± 3.55 a | 24.86 ± 4.50 a | 11.31 ± 2.30 a | 6.20 ± 0.17 | < .001 |
| Maternal uterus | 10.13 ± 2.01 a | 8.06 ± 0.48 a | 5.59 ± 0.81 a | 2.14 ± 0.42 | < .001 |
| Placenta | 5.27 ± 0.57 | 5.07 ± 0.30 | 4.64 ± 0.30 | 4.07 ± 1.35 | .034 |
| Fetus | 5.09 ± 0.75 | 4.97 ± 0.30 | 5.93 ± 0.67 b | 4.80 ± 0.33 | .012 |
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