ABSTRACT 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

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. SIMILAR CONTENT BEING VIEWED BY OTHERS PLACENTA-TROPIC VEGF MRNA LIPID NANOPARTICLES AMELIORATE MURINE

PRE-ECLAMPSIA Article 11 December 2024 INTEGRINS MEDIATE PLACENTAL EXTRACELLULAR VESICLE TRAFFICKING TO LUNG AND LIVER IN VIVO Article Open access 18 February 2021 STRUCTURAL INSIGHTS INTO

THE LYSOPHOSPHOLIPID BRAIN UPTAKE MECHANISM AND ITS INHIBITION BY SYNCYTIN-2 Article 16 June 2022 MAIN Despite continued concern about the inadvertent fetal exposure to therapeutic agents,

the last decade saw an increasing need to treat the fetus _in utero_ by administering drugs to the mother. This was largely due to a significant advancement in the diagnostic techniques(1)

to detect potentially treatable fetal pathologies in early pregnancy and to a better understanding of the natural history of these disorders(2). Encouraging results have been obtained after

maternal administration of steroid for the prevention of respiratory distress syndrome(3) and the treatment of congenital adrenal hyperplasia(4). However, transplacental fetal therapy is

restricted only to those drugs that are freely transferable across the placenta, and so attempts to treat genetically determined metabolic disorders _in utero_ have been disappointing(5–7).

The major disadvantage of this approach is difficulty in attaining therapeutic levels of a drug in the fetal circulation despite an adequate maternal concentration. This is because

distribution of a drug across the placenta depends upon _1_) pharmacologic peculiarities of the drug, _2_) differences in the maternal and fetal blood pH, _3_) the degree of protein binding,

and _4_) metabolism of the drug by the placenta. These problems are further compounded by failure to predict fetal drug levels and inadvertent maternal adverse effects to potent

medications(8, 9). Therefore, direct fetal administration of drugs by cordocentesis has been advocated to circumvent these problems(10). Nevertheless, wide use of direct fetal therapy is

limited because it is invasive and necessitates repeated administration. Hence, there is a need to develop a noninvasive drug delivery system that can effectively maintain therapeutic

concentrations of a drug in the fetus with minimal maternal side effects. Of the various carrier systems available, liposomes have been extensively investigated(11). Liposomes are

biodegradable, nontoxic, unilamellar, or multilamellar vesicles, formed from naturally occurring phospholipids, and they can entrap a wide range of drugs either in aqueous or lipid

phase(12). Liposomes have been used both in animals and humans for the treatment of infectious diseases(13, 14), metabolic disorders(15), and to minimize adverse effects of cytotoxic

drugs(16, 17). The results of these studies indicate that liposomes can effectively deliver drugs to the targeted organ(18) with minimum antigenic, pyrogenic, or other side effects(19).

Liposomes have not been used for preferential drug delivery to the fetus or the mother. In this study we hypothesize that small unilamellar liposomes can enhance placental uptake, thereby

potentiating transfer of drugs from the maternal to the fetal circulation. This assumption is based on the evidence that liposomes are avidly taken up by various cells, including

hepatocytes(20–22). To test this hypothesis we studied the transfer and uptake of small unilamellar liposomes in an _in vitro_ model of a dually perfused isolated lobule of human term

placenta by using carboxyfluorescein as a model drug. METHODS _MATERIALS_. Chromatographically pure egg phosphatidylcholine, and grade 1 dicetyl phosphate in 2:1 chloroform: methanol was

purchased from Lipid Products (Nutfield, UK). Cholesterol and stearylamine were obtained from Sigma Chemical Co. (Pool, UK). Sephadex G-25 was obtained from Pharmacia, UK. Carboxyfluorescein

from Kodak, UK. _PREPARATION OF LIPOSOMES_. Small unilamellar liposomes were prepared by the standard method that includes drying of the lipids, hydration of the lipid film, sonication, and

separation of the liposomally entrapped carboxyfluorescein from the free substance(23). Neutral liposomes were prepared with an equimolar concentration of phosphatidylcholine and

cholesterol (99 μmol). Anionic and cationic liposomes were prepared by adding dicetyl phosphate(phosphatidylcholine:cholesterol:dicetyl phosphate, 7:7:1) and

stearylamine(phosphatidylcholine:cholesterol:stearylamine, 5:5:0.05), respectively. A thin film of equimolar proportions of cholesterol and phosphatidylcholine was prepared from 2:1

chloroform and methanol suspension by rotary evaporation under reduced pressure (400-700 mm Hg) at 37 °C for 45 min. After drying the film for 30 min under a stream of nitrogen, it was

hydrated by adding 5 mL of 250 mM carboxyfluorescein solution and allowed to swell for 2 h at a temperature above the phase transition temperature (Tc) of the lipid. Liposomes of uniform

size distribution were prepared by sonicating the lipid suspension for 30 min at 4 °C under N2 (1 min burst of ultrasound followed by cooling for 30 s). The sonicated liposomal suspension

was then allowed to anneal for 2 h at room temperature and centrifuged (15 000 ×_g_ for 30 min) to separate undispersed lipid aggregates, large multilamellar liposomes, and titanium

fragments liberated from the sonicating probe. The liposomally entrapped carboxyfluorescein was separated from the free substance by gel filtration on Sephadex G-25 column (45 cm × 1 cm)

equilibrated with Tris buffer, pH 7.4. Liposomes were collected as 3-4-mL fractions of orange-colored suspensions at the end of the void volume. The free carboxyfluorescein was eluted as a

broad peak about 15 mL after the void volume. The phospholipid and cholesterol content of each liposomal preparation was analyzed. The percentage carboxyfluorescein encapsulated per mol of

lipids was calculated by measuring carboxyfluorescein latency by the formula:% latency of carboxyfluorescein = _F_2 -_F_1/_F_2 × 100, where _F_2= total carboxyfluorescein; _F_1 = free or

nonencapsulated carboxyfluorescein present. Concentration of free carboxyfluorescein per liposomal preparation was determined by suspending 10 μL of liposomal preparation in 3.9 mL of PBS.

The total concentration was measured by adding 1% Triton X-100 to the liposomal suspension to release the liposomally encapsulated carboxyfluorescein. The encapsulation efficiency of

carboxyfluorescein was expressed as the percentage of carboxyfluorescein added. The specific encapsulation efficiency of the solute entrapped by the liposomes was expressed as the percentage

per nmol of the liposomal phospholipid. Size and the number of lamellae of the liposomes was determined by negative staining with 1% ammonium molybdate under Jeol 100CX electron microscope

at low magnification followed by high magnification. _ASSESSMENT OF PERMEABILITY OF CARBOXYFLUORESCEIN CONTAINING LIPOSOMES IN BIOLOGIC MEDIUM_. Stability (in terms of carboxyfluorescein

retention) of liposomes was determined in diluted maternal and cord blood and compared with those of the PBS buffer and expressed as carboxyfluorescein retention by the liposomes. Twenty

milliliters of heparinized maternal and cord blood were collected after the delivery. One milliliter of liposomes was incubated in 5 mL of medium at 37 °C in a water bath. Samples of 0.1 mL

were taken every 30 min for 4 h in duplicate and diluted with 3.9 mL of PBS. The samples were centrifuged (1500 × _g_ for 10 min), and latency of carboxyfluorescein in the supernatant was

determined. _PLACENTAL PERFUSION TECHNIQUE_. The technique for perfusing isolated lobules of human term placenta is described in detail by Bajoria and Contractor(24). Briefly, placentas were

obtained immediately after vaginal or cesarean deliveries and perfusion of the isolated lobule was commenced within 10-15 min at 37 °C under optimal physiologic conditions of oxygenation,

pressure, flow, osmotic pressure, and acid/base status(24). Closed circuit perfusion of the fetoplacental circulations was established by cannulating the chorionic artery and vein with a

perfusion pressure of 40-50 mm Hg and a venous outflow of 6-9 mL min-1. Maternal circulation was established with an arterial pressure of 15-18 mm Hg and flow rate of 24-30 mL min-1 by

placing five cannulas in the intervillous space. Maternal and fetal perfusates had mean hematocrit values of 6 (range 4-9) and 14 (range 12-18), respectively. The maternal and fetal

circulating volume was 150-160 mL and 110-120 mL, respectively. Tissue oxygenation was maintained by oxygenating the maternal circulation with 95% oxygen and 5% carbon dioxide. Because the

resulting Po2 in the maternal (13-15 p _K_a) and the fetal circulations (4-6 p _K_a) were within physiologic limits, low Po2-mediated increased vascular tone is unlikely to occur in this

model. Diffusional viability of the perfused placenta was determined by measuring the rate of transplacental transfer of the freely diffusable marker creatinine. Only those experiments were

considered valid in which the maternal to fetal transfer of creatinine at the end of 2 h fell within the predefined range of 8-16% of the initial dose(25). Experiments were abandoned, if

fetal perfusion pressure or venous outflow were not within physiologic ranges or if the fetal circulating volume dropped by more than 2 mL due to hydrostatic fluid shift from the maternal

circuit. In five experiments, both maternal and fetal circulation were not recirculated, and 30 nM carboxyfluorescein along with 30 mg of creatinine and antipyrine were added to the maternal

perfusate only. Maternal and fetal perfusates were made from modified Tc-199 medium containing 16 g/L dextran to make perfusates isomolar and gassed with 5% CO2, 5% N2, and 95% O2 to a pH

of 7.4 at 37 °C. Samples were taken at 10-min intervals for 1 h. _EXPERIMENTAL PROTOCOL_. Just before the perfusion experiment, liposomally encapsulated carboxyfluorescein was separated from

free carboxyfluorescein by chromatography on a Sephadex G-25 column. A single bolus dose of liposome encapsulated carboxyfluorescein and 30 mg of creatinine were administered to the

maternal arterial cannula distribution head over a period of 6 min (the time required for a single maternal circulation). Two milliliters of fetal samples were taken at 15-min intervals,

whereas maternal circulation was sampled at 15 min and thereafter every 30 min for 2 h. Additional samples (0.5 mL) of maternal and fetal perfusates were taken for the estimation of pH,

Pco2, and Po2 measurements. These volumes were replaced with equal volumes of fresh perfusate. At the end of the perfusion, both circuits were drained, and their volumes were measured. The

intervillous space of the perfused placental lobule was washed with 500 mL of fresh perfusate, to remove any liposomally encapsulated drugs, or free carboxyfluorescein present in the

intervillous space. This allowed more accurate estimation of uptake of liposomes by the placenta. The perfusate draining the intervillous space was then collected in 20-mL aliquots. The

perfused placenta was dissected from the nonperfused tissue, pressure blotted to remove the excess of trapped perfusate, and stored at -20°C until weighed and analyzed. All samples were

centrifuged (3000 × _g_ for 15 min), and the supernatants were aliquoted into 0.5- and 1.5-mL volumes. Liposomal stability was determined at each sample point by measuring carboxyfluorescein

latency in a 1.5-mL aliquot. The 0.5-mL aliquot was stored at -20 °C for creatinine assay. The placental uptake of liposomes was determined after homogenizing the perfused placental tissue

in an ultra-turrax high speed homogenizer in the PBS buffer. An aliquot of the homogenate was centrifuged (3000 × _g_ for 15 min), and the carboxyfluorescein concentration was measured in

the supernatant. The concentrations of carboxyfluorescein in the maternal and fetal circulations and in the placenta were expressed as the percentage of the dose added after correction for

the background activity, the circuit volume, and the amount removed from the previous sample. Seven experiments were undertaken with 20 nM free carboxyfluorescein, and five each where

neutral liposomes or cationic liposomes was added to the maternal circulation. In nine experiments transfer of anionic liposomes was studied. _CHROMATOGRAPHY_. Two milliliters of maternal

and fetal perfusates were applied to a Sephadex G-25 column (45 × 2 cm) to fractionate liposomally encapsulated carboxyfluorescein from the free. The column was preequilibrated with Tris

buffer and eluted at room temperature with Tris-saline buffer. The elution rate was 0.63 mL min-1, and fractions of 1 mL each were collected. Each fraction was tested for carboxyfluorescein,

phosphatidylcholine, and cholesterol. _ANALYTICAL METHODS_. _Carboxyfluorescein assay_. The concentration of carboxyfluorescein was measured fluorometrically at excitation and emission wave

lengths of 490 and 520 nm, respectively, with a sensitivity of 1 nm·mL-1 and coefficient of variation of 4-7%. We prepared a standard curve of carboxyfluorescein in PBS, in maternal-fetal

perfusate, and in tissue homogenates with or without adding 1% Triton to minimize the quenching effect. Although the standard curve of carboxyfluorescein was similar in maternal and fetal

perfusates regardless of the presence of Triton, quenching effect of carboxyfluorescein in the placental tissue was approximately 10%. All values reported in this report were obtained by

reading the OD directly from the standard curve constructed using the media similar to that of the samples. _Phosphorous assay_. The phospholipid content of the liposomes was assayed by

colorimetry(26) with a sensitivity of 5μg·mL-1 and coefficient of variation of 8-10%. _Cholesterol assay_. The cholesterol content of the liposomes was assayed by colorimetry with a

sensitivity of 5 μg·mL-1 and coefficient of variation of 5-10%. _Creatinine assay_. Creatinine concentration was determined by colorimetric assay, with a coefficient of variation of 7-12%.

_DATA ANALYSIS_. All values were expressed as mean ± SEM. Two-way analysis of variance was used to compare values between groups. _p_ values <0.05 were considered significant. Equilibrium

between maternal and fetal circuits was determined when fetal/maternal ratios of the drug levels were close to unity. MAUC and FAUC of carboxyfluorescein was calculated by using the

trapezoidal rule(24). RESULTS The liposomes were unilamellar and had uniform size distribution with a mean diameter of 73.6 ± 2.8 nm. Size distribution of liposomes were comparable between

three groups (for cationic 72.1 ± 5.0, anionic 74.4± 3.6, and neutral liposomes 74.6 ± 5.7; _n_ = 4 in each group). The percentage encapsulation of carboxyfluorescein was maximum with

anionic liposomes (1.9 ± 0.2%) and minimum with cationic liposomes (1.3± 0.2%) (Table 1). Irrespective of surface charge, liposomes were stable in PBS buffer at 37 °C. In blood, the

stability of anionic liposomes declined steadily from 96.6 ± 3.0% at 60 min to 83.6± 6.9% at 4 h and were significantly more (_p_ < 0.05) leaky than the neutral (91.4 ± 3.9) and cationic

liposomes (94.6 ± 2.7) (Table 2). The transplacental transfer of carboxyfluorescein is shown in Fig. 1A and Table 3 and constitutes the control group. Maternal concentration of

carboxyfluorescein decreased slowly to 87.5 ± 1.0% at 120 min with MAUC of 10856 ± 92% dose min-1. The fetal concentration increased linearly (slope 0.30;_r_ = 0.98) to 1.3 ± 0.2% at 120

min. The FAUC and F/M ratio was 89 ± 9% dose min-1 and 0.01 ± 0.002, respectively. The placental uptake of carboxyfluorescein was 4.2 ± 1.0%. The effect of neutral liposomes on the placental

transfer and uptake of carboxyfluorescein is shown in Fig. 1B. The maternal concentration of neutral liposomal carboxyfluorescein (_p_ < 0.001) and MAUC levels (_p_ < 0.001) were

significantly less than those of the control group. The fetal concentration of carboxyfluorescein increased linearly (slope 0.21; _r_ = 0.97) and was significantly higher than the control

data (_p_ < 0.01). Similarly, FAUC (_p_ < 0.05), F/M ratio (_p_ < 0.01), and placental uptake (_p_< 0.001) of neutral liposomal carboxyfluorescein were higher than the those of

control group. The latency of carboxyfluorescein in the maternal circuit was 98.5 ± 2.1% at 15 min and 93.2 ± 2.8% at 120 min. The maternal concentration (_p_ < 0.001) and MAUC (_p_<

0.001) of anionic liposomal carboxyfluorescein was significantly less than the control data (Fig. 1C). The fetal concentration(_p_ < 0.001; slope of 0.43; _r_ = 0.98), FAUC (_p_<

0.001) and F/M ratio (_p_ < 0.001) were significantly higher than the control group. The placental uptake of anionic liposomal carboxyfluorescein was higher than the control data (_p_

< 0.001). The stability of liposomes in the maternal circulation at 97.7 ± 3.0% at 15 min and 89.9 ± 3.1% at 120 min were comparable with neutral liposomes. In contrast, the maternal

concentration (_p_ < 0.05) and MAUC levels (_p_ < 0.05) of cationic liposomal carboxyfluorescein were higher than the control data (Fig. 1D). The fetal concentration of

carboxyfluorescein (_p_ < 0.001), F/M ratio(_p_ < 0.001), and FAUC (_p_ < 0.001) were lower than those of the control group. The placental uptake of cationic liposomal

carboxyfluorescein was comparable with that of the control group. The stability of liposomes declined from 98.3 ± 2.7% at 15 min to 96.7± 3.6% at 120 min and was comparable with that of the

neutral and anionic liposomes. The cationic liposomes had significantly higher maternal concentrations(_p_ < 0.001) and MAUC (_p_ < 0.01) levels than the those of the neutral and

anionic liposomes (Table 3). The maternal levels (_p_ < 0.01) and MAUC values (_p_ < 0.001) in neutral liposomal group were significantly higher than the anionic ones. The fetal

concentrations (_p_ < 0.001) and the FAUC(_p_ < 0.001) values of cationic group were significantly less than the neutral liposomes and anionic ones (Table 3). The fetal concentration

(_p_ < 0.05) and FAUC (_p_ < 0.05) were markedly lower than that of the anionic liposomes. The tissue uptake of cationic liposomes was significantly lower than the anionic (_p_ <

0.001) and neutral liposome (_p_ < 0.001) uptake(Table 3). The placental uptake of anionic liposomes was higher than that of the neutral liposomes (_p_ < 0.01). Cationic liposomes had

a significantly lower F/M ratio than those of neutral(_p_ < 0.001) and anionic liposomes (_p_ < 0.001)(Table 3). The F/M ratio of the neutral liposomes was also significantly lower

than that of the anionic liposomes (_p_ < 0.001). The chromatogram of the maternal perfusates containing neutral liposomes showed presence of two carboxyfluorescein peaks. The first,

peak, immediately after the void volume, contained only the carboxyfluorescein-entrapped liposomes as evident from the latency, liposomal phospholipid, and the cholesterol content. The

second peak was due to free carboxyfluorescein (Fig. 2A). Of the total concentration of carboxyfluorescein present in the maternal perfusate at 120 min (73.3 ± 1.6%), 68.6 ± 2.9% was

liposomally encapsulated, whereas 6.1 ± 3.2% was free carboxyfluorescein. In the fetal perfusate (Fig. 2B), no intact liposomes was found, and there was a single peak which was due to free

carboxyfluorescein. The chromatogram of the maternal perfusate in the anionic liposomes group showed that, out of 67 ± 1.1% of total carboxyfluorescein at 120 min, 59.6 ± 4.7% was

liposomally entrapped, whereas 11.0 ± 3.6% was present in the free form. In the fetal perfusate, 2.9 ± 1.0% free carboxyfluorescein was present. Similarly, in cationic liposomal group, out

of 92 ± 1.1% of carboxyfluorescein present in the maternal circulation at 120 min, 85.0 ± 2.2% was liposomally encapsulated, whereas 6.4 ± 4.2% was present in the free form. In contrast, in

the fetal circulation, in all the three groups carboxyfluorescein was present in the free form only. DISCUSSION This study shows that small unilamellar liposomes can modulate placental

uptake and transfer of small, polar, hydrophilic molecules. Furthermore, the placental uptake of liposomes depends upon their surface charge in that neutral and anionic liposomes were taken

up more avidly than the cationic ones. Human placenta constitutes an anatomical barrier to the free passage of carboxyfluorescein from the maternal to fetal circulation. The minimal transfer

of carboxyfluorescein by the placenta is unlikely to be attributed to its molecular weight (376), because substances with similar molecular weight, such as antipyrine (188), warfarin (330),

phenytoin (275), diazepam (285), and ziduovidine (267) cross the placenta freely(27–29). It is unlikely that the lack of transport of carboxyfluorescein is a result of failure to establish

juxtaposition between maternal and fetal circulations, because in all our experiments the transport of a freely diffusable compound creatinine was similar to that obtained in previously

published perfusion experiments(24, 25). Furthermore, we have extensively characterized this model to study the transfer of drugs and macromolecules, results of which are similar to that of

_in vivo_ studies. The impermeability of the placenta to carboxyfluorescein is more likely to be due to its hydrophilicity and negative charge. The rate of transfer of polar molecules such

as chlorazepate and cimetidine are significantly lower than that of uncharged compounds with similar molecular weights because they cross the placenta by paracellular routes(24, 30). In our

study we used carboxyfluorescein as a liposomal marker because it does not _1_) cross the placenta in significant quantities,_2_) bind to liposomal membranes or tissue protein, and _3_)

undergo biotransformation. Because of these properties the clearance of carboxyfluorescein once encapsulated is expected to be similar to that of liposomes. Last, it can be easily quantified

by using simple procedures such as fluorometry. Our data show that percentage encapsulation of carboxyfluorescein by neutral and anionic liposomes was significantly higher than the cationic

liposomes. This difference could be attributed to interaction between the negatively charged carboxyfluorescein and the positively charged lipids. Liposomes, despite being stable in PBS,

were more leaky in the blood-based medium. The instability of liposomal membranes in blood is due to pore formation in the lipid bilayer by HDL, which is more marked in small unilamellar

liposomes because of irregularity in the packing of the surface lipid molecule(31, 32). Our data in accordance with others indicate that stability of liposomes depends upon their surface

charge. Anionic liposomes were more leaky than the neutral and the cationic liposomes. This difference is likely to be due to preferential binding of negatively charged lipids to unoccupied

cationic binding sites of the apoprotein molecules on LDL(33–35). Binding with other serum proteins such as cholesterol ester may also contribute to the instability of the liposomes(36–38).

In this study, stability of the liposomes in blood was maintained by using equimolar concentrations of cholesterol. Addition of cholesterol raises the phase transition temperature of the

liposomal lipids and thereby prevents binding of apoprotein and minimizes disruption of the membrane by HDL(39,40). This study suggests that liposomes do not cross the human placenta because

intact liposomes or liposomal lipids were not detected in the fetal circulation. This was in keeping with the notion that lipid-soluble substances with a molecular weight >500 do not

cross the placenta readily by simple diffusion(29). It is possible that liposomes like other endogenous macromolecules cross the placenta by an energy-dependent pathway such as

receptor-mediated endocytosis. Although no study has evaluated the mechanism of uptake of liposomes by the placenta, we consider that liposomes of 70 nm in size are unlikely to cross the

placental capillary bed. This assumption is based on the recent morphologic and physiologic evidence which indicates that human fetal capillaries have an intracellular cleft of <15 nm

size(41). Our data show that neutral and anionic liposomes significantly increase the placental uptake and thereby maternofetal transfer of carboxyfluorescein, whereas use of cationic

liposomes prevents the transplacental passage of carboxyfluorescein. The effect of surface charge of liposomes on placental transfer could be attributed to either a difference in liposomal

size or leakage of liposomally encapsulated carboxyfluorescein in the maternal circulation. These possibilities seem unlikely because our data show that _1_) stability of the liposomes was

preserved during 2 h of perfusion and was comparable among three groups of liposomes, _2_) fetal concentration of carboxyfluorescein was significantly different from the control data, and

_3_) size distribution of liposomes among three groups was comparable. It is possible that placental uptake of liposomes is depended upon the density of charge. But in this study we

evaluated the effect of only a given molar ratio of anionic or cationic lipid upon placental uptake of liposomes. However, it is possible that placental uptake can be modulated by varying

the proportion of surface charge of liposomes. Currently we are testing this hypothesis by determining the effect of different proportions of charged lipids upon the placental uptake of

anionic liposomes. Increased maternofetal transfer of carboxyfluorescein in neutral and anionic liposomes is likely to be secondary to their enchanced uptake by the placenta. Various studies

have indicated that, for a given size and lipid composition, anionic liposomes are internalized more avidly than are the neutral and the cationic ones(21, 22, 39, 40). However, the

underlying mechanism of uptake of liposomes according to their charge is not clear. It has been proposed that this may be due to more avid adsorption of phagocytic promoting factors such

asα2-macroglobulin, fibronectin, IgG, and C-reactive protein to the anionic liposomes than the cationic vesicles(42, 43). Furthermore, surface charge of the liposomes by altering the steric

arrangement of the adsorbed protein makes liposomes recognizable by various cell lines(32, 43, 44). As we used autologous blood-based perfusate, it is highly likely that liposomes will

adsorb various plasma proteins, and thereby their clearance by the placenta is likely to be similar to that by the liver. However, this study fails to elucidate the mechanism by which

liposomes are internalized by the trophoblast. We envisage that as placental uptake of liposomes depends upon their surface charge, it is possible that liposomes are internalized by an

energy-dependent pathway. Further studies using cytotrophoblast cells in culture are necessary to substantiate this hypothesis. In conclusion, our data suggest that neutral and negatively

charged small unilamellar liposomes can enhance the placental uptake and transfer of the hydrophilic, polar compound, carboxyfluorescein. If similar findings can be reproduced _in vivo,_

liposomes can be used as a model for a noninvasive drug delivery system to the fetus. ABBREVIATIONS * MAUC: maternal area under the curve * FAUC: fetal area under the curve * F/M:

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INFORMATION AUTHORS AND AFFILIATIONS * Academic Department of Obstetrics and Gynaecology, Charing Cross and Westminster Medical School, London, W6 0XG, United Kingdom Rekha Bajoria & 

Soli F Contractor Authors * Rekha Bajoria View author publications You can also search for this author inPubMed Google Scholar * Soli F Contractor View author publications You can also

search for this author inPubMed Google Scholar RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Bajoria, R., Contractor, S. Effect of Surface Charge of

Small Unilamellar Liposomes on Uptake and Transfer of Carboxyfluorescein across the Perfused Human Term Placenta. _Pediatr Res_ 42, 520–527 (1997).

https://doi.org/10.1203/00006450-199710000-00017 Download citation * Received: 12 December 1996 * Accepted: 18 June 1997 * Issue Date: 01 October 1997 * DOI:

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