Liposomal thyroxine: a noninvasive model for transplacental fetal therapy.

Drugs that cross the placenta sparingly are currently given directly to the fetus by invasive procedures. We investigated whether anionic small unilamellar (SUV) liposomes of different lipid compositions enhanced the transfer and uptake of T4 in an in vitro model of perfused human term placenta. T4-encapsulated anionic liposomes were prepared using lecithin (F-SUV) or distearoyl phosphatidylcholine (S-SUV) with cholesterol and dicetylcholine. The size distribution, encapsulation efficiency, and stability were determined in blood-based media. The transfer kinetics of free and liposomally encapsulated T4 were studied in a dually perfused isolated lobule of human term placenta, with creatinine and liposomal carboxyfluorescein as marker substances. Concentrations of T4 and rT3 were measured by RIA. T4 crossed the placenta sparingly (1.9 +/- 0.5%) because it was metabolized to rT3 (9.2 +/- 1.3%). Transplacental transfer of T4 was significantly increased by F-SUV (15.8 +/- 2.1%; P < 0.001) and S-SUV liposomes (7.1 +/- 1.2%; P < 0.001), with a concomitant decrease in fetal rT3 levels (P < 0.001). Placental uptake of F-SUV (13.5 +/- 2.0%; P < 0.001) was greater than that of S-SUV liposomes (6.7 +/- 0.8%; P < 0.001). Our data suggest that anionic liposomes increase transplacental transfer of T4. If confirmed in vivo, liposomes may provide an alternative noninvasive method of drug delivery to the fetus.

Effect of surface charge of small unilamellar liposomes on uptake and transfer of carboxyfluorescein across the perfused human term placenta.

We aim to investigate the effect of surface charge of small unilamellar liposomes on transfer and uptake of a low molecular weight, hydrophilic and polar molecule carboxyfluorescein in an in vitro model of perfused human term placenta. Carboxyfluorescein-encapsulated neutral liposomes were prepared by using an equimolar concentration of lecithin and cholesterol. Anionic and cationic liposomes were prepared by adding dicetylcholine and stearylamine, respectively. Size distribution, encapsulation efficiency, and stability of liposomes in blood-based medium were determined. The transfer kinetics of free carboxyfluorescein and liposomally encapsulated carboxyfluorescein were studied in a dually perfused isolated lobule of human term placenta. The concentration of carboxyfluorescein was measured fluorometrically. The maternal to fetal transfer and placental uptake of free carboxyfluorescein was 1.6 +/- 0.1% and 4.2 +/- 0.1% of the initial dose, respectively. This constitutes the control data. The placental transfer of carboxyfluorescein was significantly increased by neutral (2.5 +/- 0.1%; p < 0.01) and anionic liposomes (3.1 +/- 0.2%; p < 0.001), whereas cationic liposomes prevented its transfer (0.4 +/- 0.1%; p < 0.001). The placental uptake of neutral (14.9 +/- 2.3%; p < 0.001) and anionic liposomes (21.1 +/- 1.2%; p < 0.001) were significantly higher than the cationic liposomes (2.3 +/- 0.6%) and control group (p < 0.001). The placental uptake of cationic liposomes was comparable with the control data. These results indicate that placental uptake of small unilamellar liposomes depends upon their surface charge, and transfer of carboxyfluorescein is enhanced by anionic and impeded by cationic liposomes.

Effect of the size of liposomes on the transfer and uptake of carboxyfluorescein by the perfused human term placenta.

The effect of the size of liposomes on the uptake and transfer of the low molecular-weight, hydrophilic and polar molecule carboxyfluorescein has been determined across the perfused human term placenta. Carboxyfluorescein-encapsulated neutral liposomes of three different sizes were prepared from equimolar concentrations of lecithin and cholesterol. Size distribution, encapsulation efficiency and stability of liposomes in blood-based media were determined. The concentration of carboxyfluorescein was measured spectrophotometrically. The transplacental transfer and placental uptake of free carboxyfluorescein (control data) were respectively 1.9 +/- 0.2 and 5.0 +/- 0.7% of initial dose. The placental uptake and foetal concentration of carboxyfluorescein were significantly increased by small liposomes (P < 0.05), and reduced by large (0.82 +/- 0.13%; P < 0.05) and multilamellar liposomes (0.32 +/- 0.11%). There was a negative correlation between liposome size and transplacental transfer (y = -0.53 + 0.9x; r = 0.96; P < 0.001; n = 24) and placental uptake of carboxyfluorescein (y = -5.9 + 6.5x; r = 0.84; P < 0.001; n = 24). The study indicates that placental uptake and transfer rate of liposomal carboxyfluorescein were dependant upon the size of liposomes.

Endocytotic uptake of small unilamellar liposomes by human trophoblast cells in culture.

We evaluated the mechanism of uptake of carboxyfluorescein-containing small unilamellar liposomes of different surface charge by trophoblast cells in culture. Carboxyfluorescein-encapsulated neutral liposomes were prepared by using equimolar concentrations of lecithin and cholesterol. Anionic and cationic liposomes were prepared by adding dicetylphosphate and stearylamine. Trophoblast cells from human term placenta were cultured and incubated on the first day at 37 degrees C with liposome-encapsulated carboxyfluorescein or 500 nM of free carboxyfluorescein. The mechanism of uptake was determined by pre-treating the cells with metabolic inhibitors: 2 mM of sodium azide and 25 mM of deoxyglucose for 30 min. The uptake of liposomes was also evaluated both qualitatively under fluorescent microscope and quantitatively by measuring carboxyfluorescein fluorometrically. The uptake of free carboxyfluorescein and cationic liposomes was comparable. The anionic liposomes were taken up by the trophoblast cells more avidly than the neutral (13.2 +/- 1.6 versus 9.5 +/- 1.4%; P <0.01), cationic (2.9 +/- 0.4%; P <0.001) and the free carboxyfluorescein (2.1 +/- 0.9%; P <0.01). When cells were pre-treated with metabolic inhibitors, the uptake of anionic (5.9 +/- 1.8%; P <0.001) and neutral liposomes (4.0 +/- 0.8%; P <0.01) was significantly reduced, whereas uptake of cationic and free carboxyfluorescein remained unaltered. This study indicates that small unilamellar liposomes are internalized by the trophoblast cells in culture by an energy-dependent pathway; most probably by endocytosis. The neutral and anionic liposomes are internalized more avidly than cationic liposomes.

Differential binding of warfarin to maternal, foetal and non-pregnant sera and its clinical implications.

The object of this study was to determine whether differential binding of sodium warfarin in paired maternal and cord sera accounts for its adverse effects on the foetus. In-vitro binding of sodium warfarin to human serum albumin in maternal, foetal, and non-pregnant (control) subjects was determined by equilibrium dialysis at 37 degrees C. Our data suggest that at therapeutic concentrations, sodium warfarin has a single high affinity binding site on human serum albumin with an association constant of 1.65 x 10(-3) M. Serum albumin concentration in the control sera (4.42 +/- 0.08 g dL-1) was comparable with that in the cord sera (4.54 +/- 0.26 g dL-1) but was significantly higher (P < 0.01) than the maternal levels (4.03 +/- 0.21 g dL-1). Binding data indicate that the fraction of unbound warfarin in the foetal sera (6.8 +/- 1.9 g dL-1) was significantly higher than in the maternal (3.60 +/- 1.3 g dL-1; P < 0.01) and non-pregnant sera (1.96 +/- 0.6 g dL-1; P < 0.001). The maternal and foetal fractions of free warfarin were directly proportional to the concentrations of free fatty acids (y = 1268 - 110x; r = 0.93; P < 0.001), and bilirubin (y = 8.7 + 1.4x; r = 0.91; P < 0.001), respectively. This study indicates that warfarin was more strongly bound in the maternal sera than in the foetal sera; this was probably because of the competitive and allosteric effect of free fatty acid and bilirubin in the maternal and foetal sera, respectively. The clinical significance of this observation is discussed in this paper.

Comparison of the intracellular pathways of immunoglobulin-G and low density lipoprotein in cultured human term trophoblast cells.

Trophoblast cells were cultured on microporous membrane filters. After incubation at different times with gold-conjugated ligands, the cells were processed for electron microscopy. Gold particles indicating the presence of both IgG and LDL appeared in a time-dependent manner in coated pits and coated vesicles. LDL-gold appeared primarily within lysosomes whereas approximately 50% of the internalized IgG-gold appeared within vesicles (diameters ranging from 35 to 80 nm) near the basal regions of the cell. These vesicles may be the protective mechanism which prevents IgG breakdown during transcytosis across trophoblast cells, thus allowing transport of the intact molecule to the fetus.

Transfer of heparin across the human perfused placental lobule.

A system of dual perfusion of an isolated lobule of term human placenta was used as a model to study the transfer of heparin from maternal to foetal circulation. The metabolic viability of the system was assessed by measuring beta-HCG and alkaline phosphatase levels in both maternal and foetal perfusates. Creatinine and antipyrine were used as markers to determine juxtaposition of the maternal and foetal circulations. Results of this study indicate that following administration of a single bolus dose of heparin into the maternal circulation, its concentration declined slowly from 99.01 +/- 2.98 at 15 min to 97.23 +/- 4.12% and transfer of heparin in the foetal circulation was linear and increased from 0.10% +/- 0.05% at 15 min to 0.46 +/- 0.19% over a period of 120 min. The maternal (MAUC) and foetal (FAUC) concentration-time integrals were found to be 70160 +/- 1332 and 340 +/- 30 int. units min mL-1, respectively. Placental permeability of heparin and creatinine, calculated as the ratio of foetal concentration to the integral maternal-foetal concentration difference, was 8.65 x 10(-5) +/- 0.80 x 10(-5) and 0.033 +/- 0.006 mL min-1 g-1 of perfused placental weight, respectively. These data suggest that heparin was transferred from the maternal to the foetal circulation in small quantities.

Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of immunoglobulin-G (IgG) in the human placenta.

Isolated lobules of freshly delivered human term placenta were (a) subjected to an indirect immunoelectron ultracryo method in which the immunoreactivity of endogenous Immunoglobulin-G (IgG) to rabbit anti-human IgG antibody was localized with protein-A-colloidal gold and (b) extracorporeally perfused and human IgG molecules complexed to horseradish peroxidase (HRP) added to the maternal perfusate and the uptake of IgG-HRP over different perfusion durations visualized ultrastructurally by using diaminobenzidine cytochemistry. Immunoreactivity to anti-human IgG antibody was localized all along the apical plasmalemma, in apical coated and uncoated vesicles, in apical and juxtanuclear multivesicular bodies, and in basal vesicles of the syncytiotrophoblast layer of the placenta. The stroma separating the syncytiotrophoblast from the foetal endothelium as well as vesicles within the endothelium were immunoreactive. No immunoreactivity was localized in paracellular clefts of endothelia. A similar distribution of exogenous IgG-HRP was observed for the perfused placentae. When bovine IgG-HRP or HRP alone were used as control tracers no uptake was seen for the former whilst the latter was observed only in early endosomal vesicles of the syncytiotrophoblast. The pattern of localization visualized in both studies is consistent with receptor-mediated uptake of IgG by the syncytiotrophoblast and a vesicular transport of IgG across the foetal endothelium.

Vectorial transcytosis of immunoglobulin G by human term trophoblast cells in culture.

Trophoblast cells were grown on filters that allow access to apical and basal surfaces of cells. Using this experimental system, IgG transport was shown to be specific and to occur primarily in the apical to basal direction. This transport was time- and temperature-dependent, with approximately 10% of added IgG appearing on the basal side within a 60-min incubation at 37 degrees C. Other substances such as heparin were transported only minimally, whereas horseradish peroxidase was transported to the same degree in both directions. Greater than 90% of the transported IgG was precipitable by trichloroacetic acid and 81% was capable of binding to protein G. Such a rapid transport of large amounts of IgG in trophoblast cells is consistent with a receptor-mediated process of transcytosis.

Uptake and intracellular routing of peroxidase-conjugated immunoglobulin-G by the perfused human placenta.

Selected lobules of term human placenta were extracorporeally perfused and human immunoglobulin-G complexed to horseradish peroxidase (IgG-HRP) was added to the maternal perfusate. After different durations of perfusion IgG-HRP was visualised by use of diamino-benzidine cytochemistry. Within the first 10 min of perfusion IgG-HRP was found bound to microvilli and coated pits of the syncytiotrophoblast; internalisation into coated vesicles and tubulo-vesicular bodies was also observed. Subsequently, IgG-HRP was found in multivesicular bodies and by 30 min appeared in basal vesicles, the frequency of the latter event increasing with time. No routing of IgG-HRP into Golgi regions or lysosomes could be detected. by 60 min IgG-HRP was found in a few caveolae of fetal endothelium of both terminal and intermediate villi. IgG-HRP was not found in intercellular clefts of the endothelium. The pattern of uptake and routing observed suggests a receptor-mediated transcytosis of IgG-HRP across the syncytiotrophoblast and a transcellular pathway through the endothelium.

Binding and internalization of immunoglobulin G by cultured human trophoblast.

A preparation consisting of a high percentage of trophoblasts can be obtained by centrifuging a mixture of cell types from trypsinized term placental villi on a discontinuous gradient of Percoll. When cultured these cells maintain trophoblast characteristics as judged by immunocytochemical markers and beta-human chorionic gonadotrophin production. Incubation of cells with labelled human immunoglobulin G demonstrated their ability to bind and internalize this macromolecule in a time- and temperature-dependent manner.

Immunogold localisation of endogenous immunoglobulin-G in ultrathin frozen sections of the human placenta.

Endogenous immunoglobulin-G was localised in ultrathin frozen sections of human term placenta by use of an indirect immuno electron-histochemical methodology. Immunoreactivity of endogenous IgG to rabbit anti-human immunoglobulin-G antibody was visualised by use of protein-A--colloidal gold complex. Gold marked the syncytiotrophoblast in both coated and uncoated regions of the apical plasmalemma, in vesicles and multivesicular bodies, and in vesicles near the basal plasmalemma. Immunoreactivity was also seen in the interstitial space between the trophoblast and the fetal endothelial layer as well as in various types of vesicles within the endothelial cells. No immunoreactivity was seen in the intercellular clefts of the endothelium. The pattern of localisation observed is consistent with receptor-mediated uptake of immunoglobulin-G into the syncytiotrophoblast of the human placenta followed by release into the interstitial space and then vesicular transport through the endothelium.

Uptake of alpha 2-macroglobulin-trypsin complex by human placenta is mediated by a microvillous membrane receptor.

The fate of native alpha 2-macroglobulin (alpha 2M) or its trypsin complex (alpha 2M-T) was studied in the isolated dually-perfused lobule of term human placenta. [125I]-alpha 2M added to the maternal circuit was unchanged during the course of the perfusion with minimal activity becoming associated with the placental tissue. Transfer of radioactivity into the fetal circulation accounted for only 0.07 per cent of the initial dose after 2 h. In contrast, [125I]-alpha 2M-T was rapidly taken up into the placental tissue (nearly 28 per cent of the initial dose during the 2-h perfusion) and breakdown products were released into both maternal and fetal circulations. At the end of 2 h, radioactivity levels on the fetal side were 13 times higher than those found with the native protein. These indications of a classical receptor-mediated uptake and breakdown pathway were confirmed in experiments in which the acidotrophic agent chloroquine was added to the maternal circuit prior to the alpha 2M-T. In the presence of chloroquine, tissue uptake was inhibited and the subsequent release of radioactive degradation products into the fetal circuit was similar to the levels seen with alpha 2M. Incubation of term trophoblast cells at 37 degrees C with [125I]-alpha 2M-T revealed over three-fold greater cell-associated activity than was found with the native protein. In another series of experiments, a purified microvillous membrane fraction was prepared from term placentae using buffers containing 1 mM iodoacetate. In the presence of this proteolytic enzyme inhibitor, binding studies showed a single class of low affinity receptors for the alpha 2M-T complex capable of binding 4.8 +/- 1.3 (SEM) micrograms of complex per mg of membrane protein. There was no binding of the native protein.

Human placental cells in culture: a panning technique using a trophoblast-specific monoclonal antibody for cell separation.

A monoclonal antibody specific to human trophoblast has been used to separate these cells from a mixture of heterogeneous cell types following trypsinization of placental villi. A panning technique was used whereby antibody-labelled cells were incubated on a goat-anti-mouse-IgG-coated surface. The adhered cells were kept in culture for several days and assessed using three different monoclonal antibodies as cell markers. These cultures were shown to be enriched in trophoblast cells.

Characterization and specificity of lipoprotein binding to term human placental membranes.

The binding characteristics of very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoprotein fractions to a purified human term placental microvillous membrane preparation were determined. Binding of LDL was saturable with a maximal binding capacity of 270 ng LDL protein per mg of membrane protein. Scatchard analysis revealed the presence of a single population of 3.4 X 10(11) sites per mg of membrane protein and a mean affinity constant of 5.8 X 10(-9) M. Binding of VLDL was also saturable but the maximal capacity was 4.5-times greater than that of LDL. The Scatchard analysis revealed the presence of 2.1 X 10(11) binding sites and an affinity constant nearly one order of magnitude greater than that of LDL. Binding of HDL showed less tendency to saturate. Scatchard analysis showed a similar number of receptor sites to that calculated for VLDL and LDL but the affinity constant for HDL was over 100-fold less than that of VLDL. Self- and cross-inhibition studies of VLDL and LDL binding revealed that VLDL was better at blocking the binding of LDL than was LDL itself. This preferential binding of VLDL suggests that this lipoprotein fraction could be an important source of cholesterol for placental progesterone production.

Uptake and metabolism of epidermal growth factor in the perfused human placenta.

Uptake of 125I-labelled epidermal growth factor into trophoblast, and its subsequent fate, was studied in an isolated dually-perfused lobule of term human placenta. 125I-EGF added into the maternal circulation was rapidly taken up into the placental tissue where a portion was degraded and most of the breakdown products released back into the maternal circuit. At the end of the 2 h perfusion, radioactivity in the tissue accounted for 52% of the initial dose. 12.9% of the radioactivity remaining in the maternal circuit at the end of the perfusion, amounting to only 5.2% of the initial activity, could be identified as intact EGF by immunoaffinity chromatography. About 45 min after the start of the perfusion there was a sustained rise in the 125I activity in the fetal circulation accounting for 4.6% of the initial activity, and a small proportion of this (0.22% of the dose) could be immunologically characterised as EGF. In the presence of the acidotrophic agent chloroquine, there was a similar rapid clearance from the maternal circulation, which was not associated with breakdown. The tissue retention was slightly enhanced and there was very little transfer of activity into the fetal circulation.

Monoclonal antibodies to cytotrophoblast and syncytiotrophoblast of human placenta.

Mouse monoclonal antibodies were raised against plasma membranes-enriched fractions of 8-12 week placental villi. Several antibodies were obtained and three of these are described in detail: ED822 reacts with the outer membrane of the syncytiotrophoblast; ED235 reacts with the cytotrophoblast layer; ED341 reacts with the whole trophoblastic layer. Antibodies ED235 and ED341 were useful markers for identification of trophoblast cells in culture.

Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta.

The transfer of iron between the maternal and fetal circulations of an isolated perfused lobule of term human placenta was investigated using 125I-labelled or 59Fe-labelled diferric transferrin. There was negligible transplacental transfer of intact transferrin whereas nearly 4 per cent of the added 59Fe was transferred into the fetal circulation after 2 h, where it became associated with fetal transferrin. Over 20 per cent of the added 59Fe radioactivity was sequestered within the placental tissue during this period, associated with transferrin, ferritin and other uncharacterized molecules. This suggests an important role for an intracellular pool in regulating transfer. The presence of 10 mM chloroquine in the maternal circulation substantially reduced tissue accumulation of 59Fe and totally inhibited transfer to the fetus. It is concluded that the initial stages of iron transfer to the fetus involve the internalization of maternal iron-saturated transferrin bound to membrane receptors by receptor-mediated endocytosis, which can be inhibited by the drug chloroquine. Subsequently, the transplacental transfer of iron to the fetus does not involve the concomitant movement of transferrin.

Maternal to fetal movement of creatinine as a measure of perfusion efficiency and diffusional transfer in the isolated human placental lobule.

A method employing the maternal to fetal transfer of the slowly diffusing molecule creatinine in the closed-circuit dual perfusion of a term human placental lobule is described. This molecule provides an alternative to the use of antipyrine and is a useful tool in determining the overlap of the two circulations and the available exchange area in this preparation.

A monoclonal antibody based solid-phase enzyme-binding assay to measure levels of placental alkaline phosphatase in serum of women during pregnancy.

Monoclonal antibodies raised against highly purified placental alkaline phosphatase (PALP) reacted specifically with PALP and were found not to cross-react with the liver, kidney and intestinal enzymes. One of these antibodies (AP-3) was selected to develop an enzyme-binding assay to measure levels of PALP in the sera of 11 women throughout pregnancy. PALP activity rose sharply after 28 weeks of gestation reaching a peak at delivery followed by an immediate drop within 4 days to values approaching non-pregnant levels. The potential of using this assay as an index of placental function to monitor progress of a pregnancy is discussed.