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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: J Drug Target. 2014 Aug 22;22(10):935–947. doi: 10.3109/1061186X.2014.950666

A Polypeptide Drug Carrier for Maternal Delivery and Prevention of Fetal Exposure

Eric M George a, Huiling Liu b, Grant G Robinson b, Gene L Bidwell III b,c
PMCID: PMC4227969  NIHMSID: NIHMS623787  PMID: 25148609

Abstract

Background

Pregnant females are largely overlooked in drug development due to concerns for fetal health. Additionally, pregnancy is often an exclusion criterion in clinical trials, so the safety of many drugs during pregnancy is unknown.

Purpose

The goal of this study was to evaluate Elastin-like Polypeptide (ELP), a synthetic protein derived from human elastin, for maternally sequestered drug delivery. ELP is a versatile drug carrier with a long plasma half life, low immunogenicity, and the ability to be fused to nearly any small molecule or protein-based therapeutic.

Methods

We determined the pharmacokinetics, biodistribution, and fetal exposure to the ELP drug carrier using quantitative fluorescence techniques in a rat pregnancy model.

Results

After either bolus IV administration or continuous infusion over five days, ELPs accumulated strongly in the kidneys, liver, and placenta, but importantly, little to no ELPs were detectable in the fetus. Within the placenta, ELPs were localized to the chorionic plate and broadly distributed within the labyrinth, but were excluded from the fetal portion of the chorionic villi.

Conclusion

These data indicate that ELP does not cross the placenta, and they suggest that this adaptable drug delivery system is a promising platform for prevention of fetal drug exposure.

Keywords: Elastin-like Polypeptide, pregnancy, drug delivery, placental transport, preeclampsia, fetal exclusion

Introduction

Pregnant females have been a vastly overlooked patient population in drug development. Due to concerns of risk to the developing fetus, pregnancy is very often an exclusion criterion for clinical trials. As a result, the safety of drugs during pregnancy is often unknown. Other agents have exhibited toxicity in preclinical animal models, and have therefore been ruled out as therapeutics for the management of pregnancy-related disorders. Perhaps the most notable of these in the cardiovascular field are endothelin type A (ET-A) receptor antagonists. Though they show promising therapeutic effects in preclinical models of preeclampsia, ET-A receptor antagonists cause serious and even fatal effects on offspring in rodents (Alexander et al., 2001; Taniguchi and Muramatsu, 2003). Furthermore, there are disorders of pregnancy, including preeclampsia, for which there are no efficacious therapeutic interventions. Antihypertensives such as labetolol or nifedipine are sometimes prescribed with marginal success at controlling the hypertension, but there is little evidence that they affect pregnancy prolongation or the incidence of low birth weight (Fenakel et al., 1991; Sibai et al., 1992). Furthermore, these agents cross the placenta and pose some risk to the fetus (Bartels et al., 2007). Drug development for disorders of pregnancy has been nearly stagnant for decades due to concerns for fetal health and risk aversion from drug developers.

Small molecule drugs often cross the placenta, and can thus pose a threat to fetal health and development. Proteins, on the other hand, are generally too large to passively cross the placental barrier. Exceptions include immunoglobulins, which bind to the neonatal Fc receptor inside the endosomes of syncytiotrophoblasts following pinocytosis from maternal plasma and are subsequently transcytosed and released into the fetal plasma (Firan et al., 2001; Leach et al., 1996; Story et al., 1994), and transferrin, which is bound by transferrin receptors on trophoblasts and actively transported (Galbraith et al., 1980; McArdle and Morgan, 1982). This suggests that protein-fusion, using proteins that are not substrates for active placental transport, could be a viable strategy to prevent fetal drug exposure. However, little research has been performed in this area. Other high molecular weight drug carriers such as nanoparticles have been examined for placental transport and fetal uptake with mixed results. Yang et al. studied the fetal exposure to maternally administered gold nanoparticles (13 nm) in a mouse pregnancy model. They found that these particles readily transferred to the developing fetus during early gestation (prior to GD11.5), but were mostly restricted when given in late pregnancy (Yang et al., 2012). Consistent with fetal exclusion in late pregnancy, gold nanoparticles ranging from 10 to 30 nm and capped with polyethylene glyocol were not transferred to the fetal side in an ex vivo perfusion model of human placentas harvested after birth (Myllynen et al., 2008). Yamashita et al. injected 70 nm silica nanoparticles and 35 nm titanium dioxide nanoparticles intravenously into pregnant mice, and they observed these particles in the fetal liver and the fetal brain. Nanoparticle injection lead to placental structural abnormalities and reduced fetal and uterine sizes relative to control mice (Yamashita et al., 2011). Quantum dots (QD) also cross the placental barrier, though the efficiency of transfer decreases as the QD size increases (Chu et al., 2010). Fetal transfer can be reduced but not totally eliminated by capping the QDs with silica or polyethylene glycol shells. A separate study using QDs coated with mercaptopropionic acid or polyethylene glycol reported no placental transfer or teratogenicity when injected on GD6, GD13, or GD18, but the QDs did induce intraperitoneal necrosis, slightly decreased birth weight, and rarely, spots of placental and embryonic necrosis (Zalgevičienė et al., 2012). Using a different type of nanoparticle, Menjoge et al. found that PAMAM dendrimers with molecular weights of approximately 16 kDa exhibited very little transfer to the fetal side (fetal levels < 1% of maternal levels) in an ex vivo human placental transfer model (Menjoge et al., 2011). These results indicate that many nanoparticles are efficiently excluded from trans-placental transfer, especially when administered in late pregnancy. But some types of nanoparticles, including some particles with diameters as large as 70 nm, are susceptible to penetration into fetal tissue. These results demonstrate that prediction of which types of macromolecules will penetrate the placenta is not always possible a priori. The goal of this study was to determine whether a protein-based macromolecular drug carrier called Elastin-like Polypeptide (ELP) could be used for maternal drug delivery and prevention of fetal drug exposure.

ELP is a bioengineered protein with many attractive properties that make it an ideal drug carrier (Bidwell and Raucher, 2010). ELP is made of repeated units of a VPGxG pentapeptide motif, where x can be any amino acid except proline (Dan W. Urry et al., 1991). Because it is genetically encoded, the sequence of ELP is easily manipulated to change the repeat length and resulting molecule size, and the sequence can be modified to incorporate targeting peptides, reactive sites for drug attachment, or therapeutic peptides and proteins (Bidwell and Raucher, 2009; Raucher et al., 2009). ELP has previously been used for delivery of small molecule chemotherapeutics (Bidwell et al., 2007; Dreher et al., 2003; MacKay et al., 2009; Moktan et al., 2012, 2010), peptide-based therapeutics (Bidwell and Raucher, 2010, 2005; Bidwell et al., 2013, 2012, 2010; Massodi et al., 2010, 2009b, 2005), and even protein cargo (Shamji et al., 2008a, 2008b, 2007). A second advantage of ELP derives from the fact that it exhibits reversible temperature-induced aggregation. ELP is soluble below a characteristic and tunable transition temperature, but forms aggregates when the solution is raised above the transition temperature (Dan W. Urry et al., 1991; Urry et al., 1974). The aggregation is fully reversible when the solution is cooled below the polypeptide’s transition temperature. This property is leveraged in two ways. First, purification of ELP after recombinant expression is achieved by simple centrifugation at a temperature above the transition temperature, thus bypassing the need for complicated chromatographic purification protocols (Bidwell and Raucher, 2005; Meyer and Chilkoti, 1999). Second, the heat-induced aggregation allows for thermal targeting of ELP to a desired site in vivo by applying focused, externally generated hyperthermia at that site (Bidwell et al., 2013, 2012; Liu et al., 2006; Meyer et al., 2001). A third advantage of ELP for drug delivery is that it is non-immunogenic (D. W. Urry et al., 1991). The VPGxG repeat in ELP was originally derived from human elastin, and it does not induce an immune response in vivo (D. W. Urry et al., 1991). Two versions of ELP were tested in this study, one with 160 VPGxG repeats, a molecular weight of about 61 kDa, and a transition temperature of 65 °C, and a second with the same ELP modified at the N-terminus by the addition of a cell penetrating peptide (CPP) called SynB1 (Bidwell et al., 2010; Rousselle et al., 2001). We have shown previously that the addition of a CPP to ELP enhances its cellular uptake in vitro (Bidwell and Raucher, 2005; Bidwell et al., 2009, 2007; Massodi et al., 2010, 2005) and influences its biodistribution in vivo (Bidwell et al., 2013, 2012; Moktan et al., 2012). To date, most of the preclinical development of ELP has been for drug delivery applications in cancer models (Bidwell and Raucher, 2010, 2006; Bidwell et al., 2013, 2012, 2007; Dreher et al., 2003; Liu et al., 2012, 2010; MacKay et al., 2009; Massodi et al., 2009a, 2009b, 2010; Meyer et al., 2001; Moktan and Raucher, 2012; Moktan et al., 2012). This study defines the pharmacokinetics, biodistribution, placental uptake, and fetal uptake of ELP and SynB1-ELP in a preclinical pregnancy model and lays the foundation for the use of ELP to safely deliver therapeutics in pregnant patients.

Materials and Methods

Purification of Elastin-like Polypeptides

The coding sequence for ELP with the sequence (VPGxG)n where x is V, G, or A in a 1:7:8 ratio, respectively and n = 160 was cloned into the pET 25b expression vector as described previously (Bidwell and Raucher, 2005). Similarly, the coding sequence for SynB1-ELP (containing the same ELP moiety modified N-terminally with the SynB1 CPP) was cloned into pET 25b. Both polypeptides were expressed recombinantly in E. coli BLR(DE3) and purified by inverse transition cycling as described previously(Bidwell and Raucher, 2005).

Fluorescent Labeling of ELP

ELP or SynB1-ELP were covalently labeled at an engineered cysteine residue with rhodamine-5-maleimide or AlexaFluor 633 maleimide (Life Technologies) as described in (Bidwell and Raucher, 2005).

Animal Use

All animal use was approved by the Institutional Animal Care and Use Committee at the University Of Mississippi Medical Center and was carried out in accordance with the National Institutes for Health Guide for the Care and Use of Laboratory Animals. Timed pregnant Sprague Dawley rats (Charles River) were received at gestational day 11 (GD11) and acclimated to the animal housing facility.

For acute experiments, rats at GD14 were anesthetized with isoflurane and carotid catheters were placed for blood sampling. Rhodamine or AlexaFluor 633 labeled ELP or SynB1-ELP or saline control was injected by IV bolus at a dose of 100 mg/kg via the femoral vein. The injection syringe was weighed before and after injection to determine the exact dosage delivered. Blood was sampled intermittently for four hours, then the major organs, placentas, and pups were removed for fluorescence analysis. From each animal, two intact feto-amnio-placental units were rapidly frozen in liquid nitrogen for subsequent quantitative fluorescence and histological analysis.

For chronic experiments, rats at GD14 were anesthetized with isoflurane, a dose of carprofen (5 mg/kg) was administered SC, and minipumps were placed in the peritoneal cavity via a small abdominal incision. The pumps delivered 30 mg/kg/day of saline or rhodamine labeled ELP or SynB1-ELP. The incision was closed with 4-0 gut suture followed by skin closure with 4-0 prolene suture. Blood was sampled at 1h, 2h, 4h, and 8h following pump implantation and twice daily thereafter by tail prick until GD19. Bacon flavored carprofen tablets (2 mg carprofen / tablet, BioServe) were fed daily for 2 days after pump implantation. On GD19, rats were again anesthetized with isoflurane, blood was drawn from the abdominal aorta, and organs, placentas, and pups were harvested for fluorescence analysis. Two feto-amnio-placental units from each animal were rapidly frozen for quantitative fluorescence and histological analysis.

Pharmacokinetic Analysis

Fluorescence intensity of plasma from both acute and chronic biodistribution experiments was determined using a Nanoquant plate and a fluorescence plate reader (Tecan). 2 μL of undiluted plasma was measured at 543 nm excitation and 575 nm emission wavelengths and a PMT gain of 90. In addition to plasma measurements, the fluorescence of standards made from the injected proteins was determined at the same fluorometer settings. Generation of standard curves from the same batch of protein that was injected allows for correction for any variation in labeling efficiency between proteins. Plasma fluorescence was corrected for autofluorescence using plasma from saline injected animals, and the corrected fluorescence values were fit to the standard curves to determine plasma ELP concentration (μg/mL). For acute experiments, plasma concentration versus time was fit to a two-compartment pharmacokinetic model, and pharmacokinetic parameters were calculated as described in (Bidwell et al., 2012).

For chronic IP infusion experiments, once steady state plasma levels were achieved (24 h and later time points), plasma data were fit linearly to determine the average plasma ELP level. All fitting was performed using GraphPad Prism.

In Vitro Dye Release

Rhodamine-labeled ELP or SynB1-ELP was incubated at a concentration of 100 μM in serum from pregnant rats for 0 – 24 h at 37 °C. An aliquot of each sample was set aside for measurement of total fluorescence, then the protein fraction was precipitated by addition of 1:1 volume of 10 % trichloroacetic acid (TCA) and separated by centrifugation for five minutes at 13,000 × g. The fluorescence of the samples before and after TCA precipitation was measured with a fluorescence plate reader and a Nanoquant plate (Tecan) using 543 nm excitation and 575 nm emission and a gain value of 75. The fluorescence of the TCA supernatant was corrected for dilution and divided by the total sample fluorescence before precipitation to determine the % free dye. Data represent the mean ± s.d. of two independent experiments.

In Vivo Protein Stability

Plasma from each sample from the pharmacokinetic experiment (5 μL) was electrophoresed on a 10% SDS-PAGE gel under non-reducing conditions, and 10 μg of the injected protein was used as a loading control. Gels were imaged with a Chemi-doc imager with transillumination and an ethidium bromide emission filter to directly visualize the rhodamine label. One gel was run for each animal in the study, and all gels were imaged with 1.0 s exposure time. Total lane intensity and intensity of all bands < 50 kDa were determined using Image J. The percentage of the total lane intensity < 50 kDa was calculated and corrected for the % < 50 kDa of the loading control. Data represent the average ± s.d. of 4 rats per group. One representative gel from each group is shown.

Ex vivo Fluorescence Imaging

Tissue levels of fluorescent proteins were determined by ex vivo whole organ fluorescence imaging using an IVIS Spectrum (Perkin Elmer). 8 placentas and their associated pups from each experimental rat were dissected from the amniotic sac and imaged in the IVIS with 535 nm excitation and 580 nm emission, field-of-view B, f stop = 2, and auto exposure. Heart, liver, kidney, spleen, lungs, and brain were imaged similarly. Standards were prepared from the injected proteins and plated 100 μL per well in a 96-well plate. The plate of standards was imaged using identical parameters. Mean fluorescence radiant efficiency of each organ or standard was determined using a closely drawn region of interest using Living Image 4.3 software (Perkin Elmer). The mean fluorescence radiant efficiency was averaged for all placentas and pups from each rat, levels were corrected for autofluorescence determined from tissues of the saline injected animals, and fluorescence data were fit to the standard curve of the injected protein. Mean standardized fluorescence intensity was calculated for all rats in each group (n = 8 placentas and pups per rat and 4 rats per group). To generate images for publication, one representative image of placentas and pups from each group was opened using Living Image’s “load as group” tool, and all images were displayed on the same scale to allow direct comparison of the fluorescence intensity among groups.

Quantitative Fluorescence Histology Analysis

Intact, frozen feto-amnio-placental units were sectioned using a cryomicrotome and placed on microscope slides (20 μm sections). Slides were dried and scanned using a slide scanner with a 543 nm excitation laser (ScanArray Express, Perkin Elmer, laser power 70%, PMT gain 85% for sections from the acute experiment and laser power 80%, PMT gain 95% for sections from the chronic experiment, scan resolution 50 μm). Standards of the injected protein were prepared by dilution into OCT cryo-embedding medium, drawn into a 1 cc syringe, frozen, and sectioned at 20 μm thickness to create disks of each standard cut at the same thickness as the tissue. Standards were scanned with identical scan settings. The mean fluorescence intensity of standards, placenta, and pups was determined using ImageJ software. The mean tissue fluorescence intensity was corrected for autofluorescence (determined from scans of tissues from saline injected animals) and for exact injection dosage in each animal, then fit to the standard curve to determine the amount of ELP in each tissue (μg ELP / g tissue). Placenta and pup levels were averaged for all animals (n = 10 – 20 sections / tissue and 4 rats per group), and the data represent the mean ± s.e.

Histological Staining and Microscopy

For whole-slide imaging of intact feto-amnio-placental units in rats injected with AlexaFluor 633-labeled ELPs, frozen tissue sections (20 μm) were fixed for 10 min with 4% paraformaldehyde, rinsed, and stained with rhodamine-phalloidin (5 U/mL in PBS + 1% bovine serum albumin (BSA), Life Technologies) for 20 min at room temperature. Slides were washed three times, dried, and scanned using a fluorescence slide scanner. A 543 nm excitation laser and a 633 nm excitation laser were used for detection of rhodamine and AlexaFluor 633, respectively, at a 10 μm resolution scan setting. Image brightness and contrast were adjusted manually using Image J to optimize the placental fluorescence and to detect the presence of any fetal fluorescence.

For microscopy, frozen tissue sections (20 μm) of feto-amnio-placental units from the acute and chronic injection of rhodamine-labeled ELP and SynB1-ELP were mounted on glass slides and fixed in −20 °C acetone for 10 min. Slides were briefly washed with PBS and blocked in 1% BSA in PBST solution for 1 hour, followed by incubation with 1:200 dilution of the primary mouse monoclonal antibody (anti-Pan cytokeratin C-11, Sigma-Aldrich) in 1% BSA/PBST at 4°C overnight. After 3 washes in PBST (5 min each), the slides were incubated with 1:100 Alexa Fluor 488-conjugated rabbit, anti-mouse secondary antibody at room temperature for 1 hour. Slides were then washed and counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) and covered with coverslips. The sections were examined using a fluorescent microscope interfaced with Meta-morph 6.3 software. Images were psuedocolored using Image J, and brightness and contrast were adjusted to optimize image clarity.

Statistical Analysis

Ex vivo whole-organ fluorescence data were analyzed using a two-way ANOVA with treatment parameters for protein (ELP or SynB1-ELP) and tissue, and post-hoc multiple comparisons were made by Bonferroni analysis. Differences between ELP and SynB1-ELP in the quantitative placental and pup histology assay were assessed using t-tests. A p value of < 0.05 was considered statistically significant. Statistics were calculated using GraphPad Prism.

Results

Acute Pharmacokinetics of ELPs in Pregnant Rats

We determined the plasma clearance pharmacokinetics of ELP and SynB1-ELP in pregnant Sprague Dawley rats on GD14. As shown in Figure 1, after IV bolus administration, both ELP and SynB1-ELP showed a similar clearance profile with a rapid distribution phase occurring in the first 60 min followed by a slower elimination phase. ELP had a slightly higher initial plasma concentration of 3,168 μg/mL than did SynB1-ELP (2,376 μg/mL), but SynB1-ELP cleared with a longer terminal half-life of 190.2 min relative to ELP (77.5 min). The data fit well to a two-compartment pharmacokinetic model, and the results of the fitting are shown in Table 1. SynB1-ELP had a faster plasma to tissue rate and a slower elimination rate than ELP, suggesting that the CPP promoted extravasation of the polypeptide and deposition within tissues. The faster plasma to tissue rate and the slower elimination rate contributed to the reduced apparent C0 and longer terminal half-life of SynB1-ELP relative to ELP.

Figure 1.

Figure 1

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Plasma Clearance of ELPs in Pregnant Rats. Rhodamine-labeled ELP or SynB1-ELP was injected IV (100 mg/kg) in pregnant rats on GD14. Plasma was sampled over time, and fluorescence measurements were fit to standard curves of the injected proteins to calculate plasma levels. Data represent the mean ± s.d. of four rats per group. Lines represent the fit to a two-compartment pharmacokinetic model.

Table 1.

ELP SynB1-ELP
Initial
Concentration		 Co (ug/mL) 3,168 2,376
Central
Compartment
Volume of
Distribution		 Vc (mL) 7.7 13.0
Plasma
Clearance		 Cl (mL·min−1) 0.11 0.08
Area Under
Curve		 AUC (μg·min·
mL−1)		 215,254.3 401,975.3
Tissue to Plasma
rate constant		 ktp (min−1) 0.023 0.023
Plasma to Tissue
rate constant		 kpt (min−1) 0.009 0.012
Elimination rate
constant		 kel (min−1) 0.015 0.006
Distribution Half
Life		 t1/2,dist (min) 18.3 18.8
Terminal Half
Life		 t1/2,term (min) 77.5 190.2
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Protein Stability and Dye Release

When using fluorescently labeled proteins, it is imperative that the label be stably bound in order to get accurate pharmacokinetic and biodistribution data. To determine the stability of the rhodamine label attached to the proteins via maleimide chemistry, we incubated the labeled protein in plasma from pregnant rats for various times at 37 °C. After incubation, all proteins were precipitated using a 1:1 mixture with 10% trichloroacetic acid, and the fluorescence of the remaining supernatant was measured and compared to the pre-precipitation fluorescence. As shown in Figure 2A, almost no label separated from the protein when incubated in rat plasma. Even after 24 h incubation, less than 2% of the dye was released from the protein. This demonstrates that the chemistry used to label these proteins is sufficient to produce a stable bond and confirms that our measurements are indeed of the labeled protein and not of released dye.

Figure 2.

Figure 2

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Dye release and protein stability in plasma. A. Rhodamine-labeled ELP and SynB1-ELP were incubated in plasma from pregnant rats for the indicated time at 37 °C. Percentage of dye release is shown for an average of two experiments, bars indicate s.d. In vivo protein stability was determined by SDS-PAGE analysis of plasma samples from the pharmacokinetic experiment (B – E). A representative gel from one animal in each group (B, ELP; D, SynB1-ELP) is shown. The numbers indicate time points, and the final lane was loaded with 10 μg of the injected protein as a loading control. The total band intensity and % <50kDa (C, ELP; E, SynB1-ELP) are shown for an average of four animals per group, bars indicate s.d.

In addition to measuring dye release in vitro, we also examined the degradation of the protein in plasma samples in vivo. Plasma from the pharmacokinetic experiment above was analyzed by SDS-PAGE using direct fluorescence imaging to detect the labeled protein. As shown in Figure 2B and D, the total protein intensity decreased over time as the protein was cleared from circulation. However, very little protein degradation was apparent in either the ELP or the SynB1-ELP plasma samples. The amount of degradation was determined by measuring the total band intensity of the entire lane versus the total intensity of all bands < 50 kDa. Plotting the total lane intensity (Figure 2 C and E) revealed a clearance curve that closely overlayed the clearance seen with direct plasma fluorescence measurement in the pharmacokinetic experiment (Figure 1). Analysis of the percentage of the band intensities at <50 kDa molecular weight revealed that very little degraded protein was present (right axis in Figure 2C and E). Even at the 4 hour time point, less than 20% of the total signal was present in these degraded bands. This analysis revealed that these proteins were quite stable in circulation. This analysis must be considered with two caveats. First, since these proteins are labeled with fluorophors only at their termini, only fragments containing the labeled terminus will be visible. Second, because small fragments will likely be cleared by renal filtration, they may be under-represented in this PAGE analysis.

Acute Biodistribution of ELPs in Pregnant Rats

Four hours after the IV bolus dose, tissues were removed to determine the biodistribution of the polypeptides and their placental deposition and fetal uptake. We first examined the tissues by ex vivo whole organ fluorescence imaging. As shown in Figure 3A, both ELP and SynB1-ELP accumulated to high levels in the placenta, but no polypeptide was detectable in the pups. Quantitation of the whole organ fluorescence relative to standards of the injected proteins revealed that the highest polypeptide levels were found in the kidney at this time point after bolus dosing, and high levels were also seen in the liver (Figure 3B). The placenta also accumulated high levels of the polypeptides, but the pups had barely detectable (ELP) or completely undetectable (SynB1-ELP) polypeptide levels relative to autofluorescence. The addition of the SynB1 peptide to ELP increased its uptake in most organs. The kidneys were the most affected, with SynB1-ELP levels reaching more than 4 times the ELP levels (p < 0.0001). SynB1-ELP also accumulated to significantly higher levels in the liver and heart (p = 0.014 and p = 0.0017, respectively). The mean placental levels of SynB1-ELP were almost two-fold higher than the ELP levels, but the difference did not reach statistical significance (p = 0.076). The only exception to the rule of organ levels increasing with the addition of the CPP was in the spleen, where SynB1-ELP levels were significantly lower than ELP levels (p = 0.00013).

Figure 3.

Figure 3

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Biodistribution, placental deposition, and fetal exposure to ELPs. A. Four hours after IV administration of rhodamine-labeled ELP or SynB1-ELP, polypeptide levels were determined in the placentas, pups, and major organs by ex vivo whole organ fluorescence imaging. A representative image of eight placentas and corresponding pups from one animal from each group is shown. B. Fluorescence intensities were quantified, corrected for autofluorescence, and fit to standards of the injected proteins. Data represent the mean ± s.e. of eight placentas and eight pups per rat and four rats per group. * Statistically significant as determined by a two-way ANOVA with post-hoc Bonferroni multiple comparisons (p < 0.05). ** Levels were not detectable over autofluorescence.

The ex vivo whole organ analysis gives a good snapshot of the polypeptide’s biodistribution and an estimate of the actual tissue polypeptide levels. However, due to differences in organ size and therefore variability in the transmission of light through the tissue, combined with the difficulty of creating appropriate standards to correctly assess the absorbance and scattering of light, this technique has a limited ability to assess absolute tissue polypeptide levels. Therefore, we also carried out quantitative fluorescence analysis of placental and pup polypeptide levels using cryosections of intact feto-amnio-placental units. By sectioning tissue and polypeptide standards to the same thickness, this technique allows for accurate quantitation of tissue polypeptide levels. As shown in Figure 4A, this analysis confirmed that both polypeptides accumulated strongly in the placenta, but no polypeptide was detectable over autofluorescence in the pups. The images in Figure 4A, all collected at the same scan settings, also indicate that SynB1-ELP accumulated at higher levels in the placenta than did ELP. The quantitative analysis revealed that ELP placental levels were approximately 50 μg/g of tissue (Figure 4B). The placental level was increased over two-fold by the addition of the SynB1 CPP (p < 0.0001). The quantitative fluorescence analysis also confirmed the fetal exclusion of both ELP and SynB1-ELP. Neither peptide was detectable in the pups using this method (Figure 4B). To further confirm fetal exclusion, we injected rats on gestational day 14 with ELP and SynB1-ELP labeled with the near-infrared dye AlexaFluor 633. In the near-infrared region, the tissue autofluorescence is greatly decreased. Four hours after injection, feto-amnio-placental units were frozen and sectioned as above, slides were stained with rhodamine-phalloidin to mark actin and make both the placenta and the pups visible, and scanned using a fluorescence slide scanner. As shown in Figure 4C, this method confirmed that both ELP and SynB1-ELP accumulated highly in the placenta, but were undetectable in the pups. (Note that the images in Figure 4C were individually exposed to maximize the placental fluorescence and detect any fetal fluorescence. Therefore, the image intensities do not accurately reflect the differences in placental levels between the two polypeptides.)

Figure 4.

Figure 4

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Quantitative Fluorescence Histology of Feto-amnio-placental Units. Frozen feto-amnio-placental units were cut to 20 μm sections, and slides were scanned with a fluorescence slide scanner. A. Representative images from each animal were collected with identical scan settings. B. Data from all slide scans were quantified relative to fluorescence standards made from the injected protein cut to the same thickness. * Statistically significant as determined by a t-test (p < 0.05). ** Levels were not detectable over autofluorescence. C. A similar analysis was conducted in rats injected with AlexaFluor 633 – labeled polypeptides. Shown are representative images individually exposed to maximize placental levels in order to search for any fetal fluorescence.

We also examined the placental tissue microscopically with a cytokeratin counterstain to detect trophoblast cells. Low magnification revealed that both ELP and SynB1-ELP accumulated highly at the chorionic plate (Figure 5A and B, solid arrows) and distributed diffusely within the labyrinth zone. Higher magnification revealed that both polypeptides accumulated in the cytoplasm of trophoblast cells. However, the interior of chorionic villi, which contain fetal blood and are detected by voids in the cytokeratin staining, contained no ELP or SynB1-ELP (open arrows in Figure 5A and B). These results confirm at the cellular level the observations from the whole-organ and cryosection imaging that the ELP-based drug carrier is capable of entering cytotrophoblasts in the placenta but is excluded from transport into fetal circulation.

Figure 5.

Figure 5

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Intra-placental Distribution of ELPs. Slides from Figure 4A were immunostained with a cytokeratin antibody to mark trophoblast cells (green), and fluorescence of the rhodamine-labeled ELP (A) and SynB1-ELP (B) was detected (red). The 40× magnification shows polypeptide accumulation at the chorionic plate (solid arrows) and in the labyrinth. The 100× magnification shows polypeptide in the cytoplasm of trophoblast cells but excluded from the fetal chorionic villi (open arrows).

Biodistribution of ELPs in Pregnant Rats after Continuous Administration

Ex vivo whole organ and quantitative histological fluorescence analysis revealed that ELP and SynB1-ELP accumulate highly in the placenta but are excluded from the fetus four hours after bolus administration on GD14. We also examined whether the fetal exclusion held after five days of continuous infusion of the polypeptides. ELP or SynB1-ELP was administered continuously from GD14 to GD19 using an IP minipump. As shown in Figure 6A, this technique lead to a steady state plasma level of the polypeptides beginning 24 h after pump implantation. At the dose used (30 mg/kg/day), the plasma levels were maintained at 33.94 μg/mL for ELP and 69.15 μg/mL for SynB1-ELP. These plasma levels are a reflection of many in vivo kinetic processes, including the rate of transport from the peritoneal fluid to the blood, the rate of extravasation from the blood to the tissues, and the plasma clearance rate. Since SynB1-ELP has a longer terminal plasma half-life than ELP, this likely explains why there is a higher steady-state level of SynB1-ELP in the plasma than ELP. These plasma concentration data also provide useful information for the formulation of dosages for future studies, in which ELP will be fused with therapeutic agents.

Figure 6.

Figure 6

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Plasma Levels and Biodistribution of ELPs after Chronic Infusion. A. Rhodamine-labeled ELP or SynB1-ELP was administered chronically by IP minipump from GD14 – GD19. Plasma was sampled throughout the experiment, and polypeptide levels were determined relative to standards of the injected protein. Data represent the mean ± s.d. of four rats per group. B. Ex vivo fluorescence imaging of eight pups and corresponding placentas from one rat from each group is shown. C. Fluorescence intensities were quantified, corrected for autofluorescence, and fit to standards of the injected proteins. Data represent the mean ± s.e. of eight placentas and eight pups per rat and four rats per group. * Statistically significant as determined by a two-way ANOVA with post-hoc Bonferroni multiple comparisons (p < 0.05). ** Levels were not detectable over autofluorescence.

We again performed ex vivo whole organ fluorescence analysis of the placentas, pups, and organs on GD19 following five days of continuous polypeptide infusion. Relative to the acute experiment, the placental levels of the polypeptides were lower, which resulted from the difference in dose (100 mg/kg in the bolus dosing versus 30 mg/kg/day in the chronic infusion). However, similar to the acute data, the polypeptides accumulated at high levels in the placenta but were undetectable over autofluorescence in the pups (Figure 6B). The kidneys still accumulated the most polypeptide, followed by the liver and the placenta (Figure 6C). Also, after chronic infusion, the effect of the CPP on the polypeptide biodistribution was much less pronounced. Only the kidneys contained significantly more SynB1-ELP than ELP (kidney levels of SynB1-ELP were increased four-fold relative ELP kidney levels, p = 0.01). This indicates that the increases seen in the tissues immediately after infusion were the result of faster tissue deposition kinetics for SynB1-ELP relative to ELP, and after chronic administration, the tissue levels of the two polypeptides eventually became equivalent (with the exception of the kidneys).

We also examined the feto-amnio-placental units from the chronically infused rats by quantitative fluorescence histology. As shown in Figure 7A, both ELP and SynB1-ELP were present at high levels in the placenta and in the chorionic membranes. Because the laser power and PMT gain were set higher in this experiment relative to the acute experiment, the fetal autofluorescence is more evident. But even with this higher PMT gain, the polypeptides were not readily detectable in the fetus. Quantitative analysis revealed that placental polypeptide levels were about 17 – 22 μg/g of tissue for both ELP and SynB1-ELP (Figure 7B). Pup levels were barely detectable over autofluorescence for ELP (about 3.5 μg/g, and all detection came from the fetal liver which is highly autofluorescent) and completely undetectable for SynB1-ELP. This finding is significant because CPPs have been shown to mediate uptake across plasma membranes in many systems (Heitz et al., 2009), and SynB1 has even been shown to deliver some small molecule cargo types across the blood brain barrier (Rousselle et al., 2001). However, even after five days of continuous exposure, the SynB1-ELP polypeptide did not penetrate the placental barrier.

Figure 7.

Figure 7

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Quantitative Fluorescence Histology and Intra-placental Distribution of ELPs after Chronic Infusion. A. Intact feto-amnio-placental units were sectioned and analyzed by quantitative fluorescence histology as described above. A representative section of one slide from each group is shown. B. Data from all slide scans were quantified relative to fluorescence standards made from the injected protein cut to the same thickness. ** Levels were not detectable over autofluorescence. C and D. Intra-placental distribution of ELP (red, C) and SynB1-ELP (red, D) was determined following immunostaining for cytokeratin (green).

Microscopic analysis of the placentas revealed that both polypeptides accumulated in the cytoplasm of trophoblasts. However, similar to the acute exposure results, both ELP and SynB1-ELP were excluded from the fetal side of the chorionic villi (Figure 7C). These results indicate that ELP can be used to deliver therapeutic agents to the placenta, and that the carrier can even penetrate into placental trophoblasts to deliver therapeutics to intracellular targets. However, therapeutics fused to ELP or SynB1-ELP will not cross the placental barrier and reach the fetal circulation.

Discussion

One of the most vexing problems in the development of therapeutic agents for the pregnant patient is ensuring restricted fetal exposure to the agent, or in determining that the agent has no adverse effects on fetal development. This is especially true in obstetrical complications, one of the most common of which is hypertension, which affects approximately 10% of all pregnancies (“Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy,” 2000). Concerns for fetal outcome have significantly hampered development of therapeutics for the most common complications of pregnancy, including preeclampsia (Podymow and August, 2008). A number of commonly utilized antihypertensive agents have not been fully studied in pregnant populations. Others, such as ACE-I inhibitors, cause adverse fetal outcomes and death (Lambot et al., 2001). While in most cases severe hypertension can be ameliorated to acceptable levels to allow for extension of the pregnancy to near term, early induction of labor is often the outcome, making preeclampsia a leading cause of premature birth and maternal/fetal perinatal morbidity and mortality (Roberts et al., 2003). Several potential therapies, such as endothelin-1 type A receptor antagonists, have been suggested from preclinical studies, but the known teratogenic effects have prohibited their application in the clinical population (George and Granger, 2011a). The dearth of new therapeutic options for the treatment of preeclampsia that are safe for the developing fetus is a significant inadequacy in obstetrical therapeutics.

Here, we report a novel peptide polymer-based delivery system for the safe, maternally sequestered delivery of therapeutics during pregnancy. Acute administration of ELP with or without the cell penetrating peptide SynB1 demonstrated similar plasma kinetics, though the SynB1-tagged polypeptide exhibited both a faster extravasation into the tissues in the distribution phase as well as a slower elimination rate in the elimination phase. These data can also be contrasted with a previous study in which the pharmacokinetics of similar ELP and SynB1-ELP polypeptides were determined in non-pregnant female Sprague Dawley rats of about the same age (Bidwell et al., 2013). In that study, we determined terminal half-lives of 243 and 278 minutes for ELP and SynB1-ELP, respectively. The biodistribution in the non-pregnant rats was similar, with strong accumulation in the kidneys and liver. The significantly longer half-lives in non-pregnant rats are consistent with the expected increase in drug elimination rate during pregnancy. Future work will test the effects of modifying the ELP size on the pharmacokinetics, placental deposition, and fetal transfer in order to optimize delivery to the target maternal organs while still preventing fetal exposure.

Most significantly, when the acute biodistribution of the two peptides was examined, it became readily clear that ELP with or without the SynB1 CPP was unable to significantly cross the maternal/fetal interface and was restricted to the maternal compartment. When the placenta was examined histologically, both ELP and SnyB1-ELP were found ubiquitously in the placental tissue, but were excluded from the fetal-derived placental villi, showing that the interface between the two circulations was the effective barrier of the molecules (Figure 5). Highest concentrations of both polypeptides were observed in the maternal kidney, liver, and placenta, with SynB1 increasing tissue levels in most of these organs. It is significant for potential therapeutics for preeclampsia that both the kidneys and placenta exhibit significant accumulation of the carriers. The placenta itself is the source of many of the pathogenic factors known to cause the maternal symptoms of preeclampsia, and the changes in blood pressure seen in the disorder are linked to decreased renal function and glomerular endotheliosis (Jeyabalan and Conrad, 2007; Moran et al., 2003). These organs, therefore, are the most likely candidates for intervention in the preeclampsia patient. The fetal exclusion of ELP even held after longer chronic exposures. When ELP or SynB1-ELP were administered chronically from GD14-19 (to mimic treatment throughout the final trimester of pregnancy as would be appropriate for preeclampsia therapy), we saw distributions quite similar to the acute experiments. The pattern of tissue biodistribution was similar to that seen in the acute experiments, with accumulation in the kidney, liver, and placenta but undetectable or barely detectable in the pups (Figure 6C). These data indicated that even in the setting of prolonged administration of ELP or SynB1-ELP as would be done for preeclampsia therapy, significant fetal exposure is unlikely to occur.

Our current focus is to utilize the ELP drug delivery system for delivery of therapeutics to treat preeclampsia. Preeclampsia is marked by new-onset hypertension, proteinuria, and maternal vascular dysfunction. If left unchecked, eclampsia can result, leading to maternal grand mal seizures, and in extreme cases, death. While the maternal hypertension is a significant health concern, it is not entirely clear that antihypertensive therapy per se is desirable. Preeclampsia is now thought to result from hypoperfusion and chronic ischemia of the placenta, which in turn secretes pathogenic factors into the maternal blood stream such as the VEGF antagonist sFlt-1 and inflammatory cytokines (George and Granger, 2011b). Lowered maternal pressure conceivably could lower placental perfusion even further and significantly worsen the effects of the disease. One potential therapeutic approach, then, is to directly target these placental-derived factors to prevent worsening of the maternal syndrome. However, ensuring fetal safety of new therapeutics remains a primary concern. The ELP-based delivery system described here could provide a safe carrier with which to administer new therapeutics, (either peptide or small molecule-based) to the preeclamptic patient with significantly reduced risk of causing fetal harm. Our current work is focused on development of ELP-fused protein and peptide-based antagonists of s-Flt function or inflammatory cytokine production. In addition to the management of preeclampsia, a vast array of contraindicated therapies for a spectrum of disorders could also be safely delivered in the pregnant patient. The data here support the utility of ELP conjugation as a method of maternally sequestering these potentially harmful agents. Future work testing ELP transport in human placental tissue ex vivo and assessing efficacy of ELP-conjugated therapeutics in preclinical models of pregnancy disorders such as preeclampsia should prove enlightening.

Conclusion

We have tested the protein based drug carrier ELP to determine its distribution during pregnancy in a rat model. We found that ELP can be used to target drugs to the maternal kidneys, liver, or placenta, but that little to no ELP crosses the placenta and reaches the developing fetus. ELP is a versatile drug carrier that is being developed for delivering drugs for many diseases, from cancer to cardiovascular disease. This work demonstrates that ELP can be used as a carrier to prevent placental transfer of drugs. Therefore, ELP fusion could transform drugs that are currently unsafe, or whose safety is unknown, into a formulation that is safe for use during pregnancy. Additionally, ELP fusion could allow for the development of new therapeutics for disorders of pregnancy for which we currently have no therapies.

Footnotes

Declaration of Interest.

Direct funding for this work was provided by American Heart Association grant 13SDG16490006, NIH NHLBI grant R01HL121527, and startup funds from the University of Mississippi Medical Center to G.L.B. Partial salary support for E.G. was provided by NIH grant R00HL116774. Ex vivo specimen imaging was partially supported by the Animal Imaging Core Facility of the University of Mississippi Medical Center. G.L.B. is owner of Leflore Technologies, LLC, a private company working to develop ELP-based therapies for various diseases.

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