Objective
Indomethacin (IND) is a prostaglandin production inhibitor that reduces uterine contractions, but crosses the placenta leading to adverse fetal effects. Liposomes (LIP) are nanoscale systems clinically used to preferentially deliver a drug to the tissue of interest and simultaneously prevent distribution to unwanted locations. Our objective was to determine whether LIP could prevent the transfer of IND across the placenta to the fetus while preserving its pharmacological activity.
Study Design
Multilamellar LIP were designed with a 150- to 200-nm size, fluorescently labeled, and loaded with IND. Timed pregnant CD1 mice (n = 6/group) on gestational day 18 were administered LIP, LIP-IND (1 mg IND/kg), or saline (SAL) via tail vein injection, or IND (1 mg/kg) via oral gavage. After 4 hours, the uterus, placenta, and fetuses were retrieved. LIP levels were visualized using fluorescent microscopy and quantitatively assessed by National Institutes of Health image processing software. LIP brightness values (mean ± SEM) in arbitrary units (AU) were normalized to the autofluorescence of the same tissue (as measured in SAL group). IND and prostaglandin E2 levels were assessed using liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay, respectively.
Results
The qualitative analysis of LIP distribution revealed that the system was primarily confined within the uterus, minimally detected within the placenta, and absent in the fetus. LIP fluorescence was greater in the uterus compared to placenta and fetus (uterus 15.3 ± 5.4 AU vs placenta 3.0 ± 3.5 AU vs fetus 4.4 ± 2.5 AU; P = .009). LIP-IND resulted in a 7.6-fold reduction in the IND levels in the fetus compared to IND alone (LIP-IND 10.7 ± 17.1 ng/g vs IND 81.3 ± 24.7 ng/g; P = .041). Prostaglandin E2 levels were significantly reduced in the uterus of animals given LIP-IND and IND compared to LIP and SAL.
Conclusion
LIP localized within the uterus and did not cross the placenta to the fetus. IND within the fetus was reduced 7.6-fold while encapsulated within the LIP and the pharmacologic effects of IND were maintained. Thus, LIP provide a novel therapeutic approach to correct the primary clinical limitation of IND by reducing placental passage to the fetus.
Prematurity is a leading cause of perinatal morbidity and mortality affecting 12% of approximately 4 million deliveries in the United States annually. Very few drugs have been proven to be effective in the treatment of preterm labor to improve neonatal outcomes. Unfortunately, tocolytic modalities that have shown to be effective are often limited in their use due to associated toxicities to the fetus.
Indomethacin (IND) is a commonly used tocolytic agent from the nonsteroidal antiinflammatory drugs family. The role of prostaglandins in the initiation of labor is well documented. IND is a nonselective cyclooxygenase, or prostaglandin-endoperoxide synthetase, inhibitor that suppresses the production of prostaglandins in the uterus and other locations, thus, inhibiting preterm labor. Numerous randomized controlled trials comparing IND to either placebo or other tocolytic agents have shown a significant delay in delivery of up to 48 hours in 90-94% and up to 7 days in 75% of cases. The clinical use of IND has been tempered by concerns over fetal and neonatal complications related to its ability to cross the placenta. Fetal exposure to IND is associated with the development of antenatal closure of the ductus arteriosus, antenatal oligohydramnios, postnatal persistent patent ductus arteriosus, necrotizing enterocolitis, intraventricular hemorrhage, and postnatal renal failure.
Nanomedicine is a multidisciplinary field of science, amalgamating notions of medicine and nanotechnology with the overall goal of a precise control over the processes in biology that occur on the micron/submicron scale. In medicine, challenging and unmet needs shape the opportunities to develop novel nanotechnology-enabled approaches. The venue of vectoring the drugs preferentially to the disease loci and, thus, increasing the efficacy and reducing associated toxicities and adverse reactions is one of the great promises of nanomedicine over the traditional molecular therapeutics, which equally distribute to healthy tissues in the body as well as to the target location.
Since the late 1990s, nanovectors such as liposomes (LIP) have been clinically used to enhance drug delivery to tumor sites and reduce drug side effects of chemotherapeutic and antimicrobial therapies. LIP, phospholipid based nanovesicles, are biodegradable and pose no concern for toxicity. The evolution of translating preclinical studies into patient care is best illustrated by the application of LIP for the treatment of gynecologic ovarian cancer. LIP doxorubicin (approved by the Food and Drug Administration in 1997) is a common alternative to traditional doxorubicin.
LIP have also been studied in pregnancy and have been shown to inhibit the transplacental passage of intravenous inulin, riboflavin, methotrexate, penicillin, valproic acid, and hemoglobin in human placental perfusion models. As an example, the levels of an antiepileptic drug, valproic acid delivered from LIP in the human placenta perfusion model, was reduced by 32% in the fetal circulation and 57% in the placental tissue compared to the maternal circulation. Encapsulation in cationic LIP of warfarin, a teratogenic anticoagulant drug, reduced passage to the fetus by 57-66%. Thus, LIP that are >100 nm in size, cationic (positively charged), and multilamellar (multiple layers of lipids) tend to decrease the amount of medication transfer across the placenta.
The objective of our study was to determine whether LIP prevent the transfer of IND across the placenta to the fetus in a pregnant mouse model.
Materials and Methods
LIP design and fabrication
LIP and IND-LIP were prepared by lipid hydration-extrusion method. First, the lipids were dissolved in 3 mL ethanol at the following concentrations: 107.5 mg soy bean phosphatidylcholine (Lipoid S100; Lipoid, Ludwigshafen, Germany) and 9 mg cholesterol (Sigma-Aldrich, St. Louis, MO). To fluorescently label LIP, fluorescent phospholipid Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethyl-ammonium salt (rhodamine-dihexadecanoyl-phosphatidyl-ethanolamine; Invitrogen, Carlsad, CA), 2% of the total lipid was incorporated to LIP and LIP-IND. For LIP-IND, 4.5 mg of IND (Sigma-Aldrich) was added to the above ethanolic mixture. A thin film formed by evaporating the solvent for 30 minutes, 41°C at 150 rpm using rotary evaporator (Rotavapor, Buchi, Switzerland). The film was rehydrated with 5 mL phosphate buffer saline pH 7.2. LIP were extruded 10 times using each of the following 800-, 400-, and 200-nm Nuclepore Track-Etch Membrane (Whatman, Little Chalfont, UK) filters with Lipex Biomembrane extruder (Vancouver, British Columbia, Canada). Levels of IND in the LIP were assessed following ultracentrifugation using high-performance liquid chromatography (HPLC).
HPLC method for IND detection in the formulation
IND was analyzed by isocratic detection using ultraviolet diode-array HPLC system Column Hitachi Elite LaChrom (Hitachi, Chiyoda, Tokyo, Japan), Column oven L-2300, Autosampler L-2200, Diode Array Detector L-2455, Pump L-2130 Hitachi D-2000 Elite v3.0 software. Kinetex 2.6μ XB-C18 100Ǻ (100 × 4.6 mm; Phenomenex, Torrance, CA) column was used for the separation at 237 nm. Chromatography was performed using an isocratic elution with mobile phase composed of 0.2% phosphoric acid in acetonitrile at a flow rate of 0.6 mL/min with average retention time of 7.2 minutes.
Characterization of LIP and LIP-IND
The size and zeta potential of the LIP were assessed by dynamic light scattering technique using Zetasizer instrument (Malvern, Westborough, MA). Five separately prepared batches of LIP and LIP-IND were analyzed in triplicates each. The morphology and structure of LIP were observed by scanning electron microscopy as previously described.
In vivo study in pregnant rodents
A pregnant mouse model was used to assess the biodistribution of IND and LIP-IND within the maternal uterine, placental, and fetal compartments. Timed pregnant CD1 mice (Charles River) were obtained on gestational day 13. Mice 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 in accordance with the protocol AWC #HSC-AWC-13-154 approved in January 2014 by the University of Texas Health Science Center at Houston Animal Welfare Committee.
On gestational day 18, animals (n = 6 per group) were administered 0.1 mL of LIP, LIP-IND, or saline (SAL) via tail vein injection or 1 mL of IND by gavage. When the drug was used (LIP-IND and IND groups), the dose of IND was 1 mg/kg. After 4 hours, laparotomy was performed after carbon-dioxide inhalation euthanasia and the maternal uteri, placentas, and fetuses were retrieved, weighed, and processed for the analysis of LIP, IND, and prostaglandin E2 (PGE2) by fluorescent microscopy/ImageJ, HPLC-tandem mass spectrometry and enzyme-linked immunosorbent assay, respectively, as described below. The onset of action of LIP-IND is unknown when administered intravenously. However, IND’s onset of action is 2-3 hours when given orally to human beings and rodents, and is 4-5 hours when encapsulated with LIP and administered intraperitoneally. Based on these prior studies, 4 hours was chosen as the period of exposure.
Evaluation of IND-LIP distribution
Tissue localization of LIP was qualitatively assessed using fluorescence microscopy identifying the absence or presence of LIP (tagged with fluorescent dye as previously described) within each tissue. For this analysis, the excised tissues were placed in cryo-molds, embedded in the cryo-preserving media (optimum cutting temperature medium), and immediately frozen using liquid nitrogen. The blocks were stored in –80°C until sectioning using cryo-microtome. During mounting on the slides, the tissue slices were stained with 4′,6-diamidino-2-phenylindole fluorescent stain to identify nuclear structures of cells. Images were taken with the BX51 fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan) using filters for 4′,6-diamidino-2-phenylindole and Cy3 at ×100 magnification. Quantitative biodistribution of LIP was determined using National Institutes of Health image processing software (ImageJ). LIP brightness values (mean ± SEM) in arbitrary units were recorded and normalized to the autofluorescence of the same tissue (as measured in SAL group).
Evaluation of IND levels in tissues
IND levels were measured in the LIP-IND (n = 5) and IND groups (n = 5), and normalized to the tissues of animals not treated with the drug (LIP, n = 3 and SAL, n = 3). The concentrations were determined within University of Texas Health Science Center at Houston core laboratory. Briefly, uterine, placental, and fetal tissue were homogenized in 3 mL of methanol and then centrifuged at 3500 rpm for 5 minutes. The supernatant was diluted 50-fold with methanol-water (1:1 vol/vol). Then, 50 μL of the diluted supernatant were used to measure IND levels quantitatively using liquid chromatography-tandem mass spectrometric method. IND concentrations were determined in ng/mL. IND levels were further normalized per organ weight (ng/g) and reported as mean ± SEM.
Evaluation of IND pharmacological activity
Since the pharmacological action of IND involves the inhibition of prostaglandin production by cyclooxygenase, PGE2 levels were measured in uterine tissue using enzyme-linked immunosorbent assay (Enzo Life Sciences, Farmingdale, NY). Briefly, a monoclonal antibody to PGE2 was used to bind, in a competitive manner, the PGE2 in the sample. After simultaneous incubation at 4°C, the excess reagents were washed away and substrate added. After incubation at 37°C, the enzyme reaction was stopped and the yellow color generated a measure on a microplate reader at 405 nm. The intensity of the bound yellow color is inversely proportional to the concentration of PGE2 in either standards or samples. The measured optical density used to calculate the concentration of PGE2 was expressed as pg/mL and reported as the mean ± SEM. Uterine contractions in the pregnant mouse were not measured in this study.
Statistical analysis
LIP fluorescence, IND, and PGE2 levels were compared using Kruskal-Wallis 1-way analysis of variance (nonparametric analysis) followed by Tukey post-hoc test. A P value of < .05 was considered significant.
Results
To prevent the placental passage of IND to the fetus and related toxicities, we have designed IND-LIP. Characterizations of the LIP by dynamic light scattering and scanning electron microscopy have shown that the nanoparticles appear as uniform, spherical vesicles of ∼150-170 diameter ( Figure 1 ). When the size distribution of 5 separately prepared batches of IND and LIP-IND was evaluated, the average values were 159.8 ± 1.1 nm (polydispersity index [PDI] <0.083) for LIP and 164.4 ± 4.7 (PDI 0.069) for LIP-IND. The low PDI values (<0.1) point towards the homogeneity of the formed phospholipid nanovesicles. Quantitative analysis of the drug revealed that IND encapsulation efficiency in the LIP was 93%. To enable the biodistribution in the tissues analysis, LIP and IND-LIP were tagged with red fluorescent dye.
The qualitative (red fluorescence) assessment of LIP-IND distribution revealed that the system was primarily confined within the uterus, minimally detected within the placenta, and absent in the fetus as shown in Figure 2 . Quantitatively, the LIP-IND brightness values were significantly higher in the uterus of animals given LIP compared to placenta and fetus, (uterus 15.3 ± 5.4 vs placenta 3.0 ± 3.5 vs fetus 4.4 ± 2.5; P = .009). LIP-IND system resulted in significantly lower IND levels in the fetus compared to IND alone (LIP-IND 10.7 ± 17.1 ng/g vs IND 81.3 ± 24.7 ng/g; P = .041) as described in the Table and depicted in Figure 3 .
| Variable | Uterus a | Placenta a | Fetus b |
|---|---|---|---|
| IND | 236.7 ± 21.6 | 649.7 ± 78.4 | 81.3 ± 24.7 |
| LIP-IND | 318.7 ± 54.2 | 937.8 ± 513.0 | 10.7 ± 17.1 |
a Nonsignificant (IND vs LIP-IND)
To evaluate the pharmacological effect of IND administered from LIP-IND, the levels of PGE2 were detected in the uterus. PGE2 levels were significantly reduced in the uterus of animals given LIP-IND and IND compared to LIP and SAL (LIP-IND 457.5 ± 64.0 pg/mL vs IND 493.3 ± 89.0 pg/mL vs LIP 1066.0 ± 171.0 pg/mL vs SAL 1142.0 ± 52.0 pg/mL; P = .0001) as described in Figure 4 .
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