Transport and biodistribution of dendrimers across human fetal membranes: implications for intravaginal administration of dendrimer-drug conjugates

Abstract

Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly (amidoamine)) dendrimers, across human fetal membrane (using a side by side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size approximately 400 Da) and fluorophore-tagged G(4)-PAMAM dendrimers (approximately 16 kDa). The fluorophore-tagged G(4)-PAMAM dendrimers were synthesized and characterized using (1)H NMR, MALDI TOF MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a 5 h period, the dendrimer transport across all the three membranes was less than <3%, whereas the transport of FITC was relatively fast with as much as 49% transport across the amnion. The permeability of FITC (7.9 x 10(-7) cm(2)/s) through the chorioamnion was 7-fold higher than that of the dendrimer (5.8 x 10(-8) cm(2)/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5-4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be restricted across the human fetal membranes when administered topically by intravaginal route, suggesting new ways of selectively delivering therapeutics to the mother without affecting the fetus.

Scheme 1

The schematic representation for the synthesis of fluorescently labeled G₄-PAMAM-dendrimers; G4-PAMAM-O-GABA-NH-FITC (1) and G4-PAMAM-O-GABA-NH-Alexa (2).

Fig. 1

MALDI TOF MS spectra for G4-PAMAM-O-GABA-Boc (5) (Mw = 15,960 Da), G4-PAMAM-O-GABA-NH₂ (6) (Mw = 14,949 Da), G4-PAMAM-O-GABA-NH-FITC (1) (Mw =15,805 Da) and G4-PAMAM-O-GABA-NH-Alexa (2) (Mw = 16065 Da) showing the corresponding mass.

Fig. 2

HPLC chromatograms for G4-PAMAM-O-GABA-NH₂ (6) (UV channel), G4-PAMAM-O-GABA-NH-FITC (1) (Fluorescent channel) and G4-PAMAM-O-GABA-NH-Alexa 488 (2) (Fluorescent channel). The retention time of G4-PAMAM-O-GABA-NH₂ is 16.2 min and the FITC and Alexa tagged G4-PAMAM-O-GABA-NH₂ show a peak appearing at 17.5 and 15.5 min respectively.

Fig. 3

(A) The transport of G₄-PAMAM-O-GABA-NH-FITC (D-FITC) and FITC (unconjugated) across the fetal membrane comprising amnion and chorion together over 30h in the side by side diffusion chamber. (B) The FITC shows a rapid transport across the membrane in 5 h (~20%), while the dendrimers show negligible transport of ≤ 3 % in 5 h. The concentrations of D-FITC studied were 0.6 mg/mL and 3 mg/mL. The concentration of FITC was 0.3 mg/mL.

Fig. 4

(A) The transport of G₄-PAMAM-O-GABA-NH-FITC (D-FITC) and FITC (unconjugated) across the chorion stripped off fetal membrane. The amnion was placed in the side by side diffusion chamber over 30 h to study the transport of dendrimers. (B) The FITC shows a rapid transport across the membrane (50 %) in 5 h, while the dendrimers show negligible transport of ≤ 3% in 5 h. The concentrations of D-FITC studied were 0.6 mg/mL and 3 mg/mL. The concentration of FITC was 0.3 mg/mL.

Fig. 5

(A) The transport of G₄-PAMAM-O-GABA-NH-FITC (D-FITC) and FITC (unconjugated) across the amnion stripped off fetal membrane. The chorion was placed in the side by side diffusion chamber over 30 h to study the transport of dendrimers. (B) The FITC shows a rapid transport across the membrane (~25 %) in 5 h, while the dendrimers show negligible transport of ≤ 3 % in 5 h. The concentrations of D-FITC studied were 0.6 mg/mL and 3 mg/mL. The concentration of FITC was 0.3 mg/mL.

Fig. 6

Permeability coefficient for dendrimer measured across the (A) chorioamnion (B) amnion and (C) chorion. The concentrations of G₄-PAMAM-O-GABA-NH-FITC (D-FITC) studied were 0.6 mg/mL and 3 mg/mL. The permeability coefficient of 0.6 mg/mL and 3mg/mL D-FITC through (A) chorioamnion was 7.5 × 10^-8 and 5.8 × 10^-8 respectively (B) amnion was 1.86 × 10^-8 and 2.08 × 10^-7 and (C) chorion was 2.94 × 10^-8 and 2.94 × 10^-8 cm²/s

Fig. 7

Permeability coefficient for FITC (unconjugated) measured across the chorioamnion, amnion and chorion. The concentration of FITC was 0.3 mg/mL. The permeability coefficient of FITC through chorioamnion was 7.93 × 10^-7, amnion was 2.26 × 10^-6 and chorion was 1.32 × 10^-6 cm²/s.

Fig. 8A

The H and E stained human chorioamniotic (fetal) membrane. AE = amniotic epithelium, AM= amniotic mesoderm, CM=chorionic mesoderm, CT=chorionic trophoblast, DE=decidua comprising the stromal cells. For the transmembrane study the amniotic epithelium was placed facing the receptor cell to study the transport of dendrimer from maternal side (extra-amniotic cavity) to the fetal side.

Fig. 8B

The human chorioamniotic (fetal) membrane showing the nuclei stained blue with DAPI (control membrane without the treatment with G₄-PAMAM-O-GABA-NH-Alexa (D-alexa) (20x). The negative controls rabbit isotype and mouse isotype replaced the primary antibodies. AE = amniotic epithelium, CAM = chorioamniotic mesoderm, CT = chorionic trophoblast, DE = decidua comprising the stromal cells. For the transmembrane study the amniotic epithelium was placed facing the receptor cell to study the transport of dendrimer from maternal side (extra-amniotic cavity) to the fetal side.

Fig. 9

Transmembrane transport of G₄-PAMAM-O-GABA-NH-Alexa (D-alexa) across the human fetal membrane at different time points (30 min, 1, 2, 2.5, 3, 3.5 and 4 h respectively) (20x). The nuclei are stained as blue (DAPI), the trophoblast cells in the chorion region are stained cytokeratin positive (red) and the stromal cells in the decidua are stained vimentin positive (magenta). The D-alexa (green) can be seen advancing through the different regions (the different regions are marked in the control membrane shown in bottom panel). At initial time points (30 min to 2 h) the dendrimer is seen in mostly in the decidua and stromal cells and at time points 3 to 4 h the dendrimers seem to diffuse into the chorionic trophoblast region. The image without cytokeratin and vimentin shows the diffusion of dendrimer throughout the decidua and trophoblast cells (4 h, bottom panel, center). AE = amniotic epithelium, CAM = chorioamniotic mesoderm, CT = chorionic trophoblast, DE = decidua comprising the stromal cells. For the transmembrane study the amniotic epithelium was placed facing the receptor cell to study the transport of dendrimer from maternal side (extra-amniotic cavity) to the fetal side.

Fig. 10a

The colocalization images for the G₄-PAMAM-O-GABA-NH-Alexa (D-alexa) in the decidual stromal cells at 4 h. The stromal cells are vimentin positive (magenta) and the nuclei of all the cells are stained blue. The D-alexa is seen in green. The internalization of D-alexa in the nuclei and cytoplasm of stromal cells can be seen from the merged composite image. The colocalized D-alexa with nuclei appears as cyan (63x). The arrows identify the cells showing cellular uptake of dendrimer (63x). Also the dendrimer seems largely in the interstitial regions.

Fig. 10b

The colocalization images for the G₄-PAMAM-O-GABA-NH-Alexa (D-alexa) in the chorionic trophoblast region at 4 h. The chorionic trophoblast cells are cytokeratin positive (red) and the nuclei of all the cells is stained blue. The D-alexa is seen in green. The internalization of D-alexa in the nuclei of trophoblast cells is seen from the merged composite image. The arrows identify the cells showing cellular uptake of dendrimer. The colocalized D-alexa with nuclei appears as cyan (63x). Also the bottom panel shows that dendrimer is largely in the interstitial regions (20x).