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1 These authors contributed equally to this article.
Jonathan W. Paul
Footnotes
1 These authors contributed equally to this article.
Affiliations
Mothers and Babies Research Center, School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Newcastle, AustraliaHunter Medical Research Institute, New Lambton, Australia
1 These authors contributed equally to this article.
Susan Hua
Footnotes
1 These authors contributed equally to this article.
Affiliations
School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Newcastle, AustraliaHunter Medical Research Institute, New Lambton, Australia
Mothers and Babies Research Center, School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Newcastle, AustraliaHunter Medical Research Institute, New Lambton, Australia
Mothers and Babies Research Center, School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Newcastle, AustraliaHunter Medical Research Institute, New Lambton, Australia
Mothers and Babies Research Center, School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Newcastle, AustraliaHunter Medical Research Institute, New Lambton, Australia
Hunter Medical Research Institute, New Lambton, AustraliaRobinson Research Institute and School of Medicine, University of Adelaide, Adelaide, South Australia, Australia
Mothers and Babies Research Center, School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, Newcastle, AustraliaHunter Medical Research Institute, New Lambton, AustraliaJohn Hunter Hospital, New Lambton, Australia
The ability to provide safe and effective pharmacotherapy during obstetric complications, such as preterm labor or postpartum hemorrhage, is hampered by the systemic toxicity of therapeutic agents leading to adverse side effects in the mother and fetus. Development of novel strategies to target tocolytic and uterotonic agents specifically to uterine myocytes would improve therapeutic efficacy while minimizing the risk of side effects. Ligand-targeted liposomes have emerged as a reliable and versatile platform for targeted drug delivery to specific cell types, tissues or organs.
Objective
Our objective was to develop a targeted drug delivery system for the uterus utilizing an immunoliposome platform targeting the oxytocin receptor.
Study Design
We conjugated liposomes to an antibody that recognizes an extracellular domain of the oxytocin receptor. We then examined the ability of oxytocin receptor–targeted liposomes to deliver contraction-blocking (nifedipine, salbutamol and rolipram) or contraction-enhancing (dofetilide) agents to strips of spontaneously contracting myometrial tissue in vitro (human and mouse). We evaluated the ability of oxytocin receptor–targeted liposomes to localize to uterine tissue in vivo, and assessed if targeted liposomes loaded with indomethacin were capable of preventing lipopolysaccharide-induced preterm birth in mice.
Results
Oxytocin receptor–targeted liposomes loaded with nifedipine, salbutamol or rolipram consistently abolished human myometrial contractions in vitro, while oxytocin receptor–targeted liposomes loaded with dofetilide increased contraction duration. Nontargeted control liposomes loaded with these agents had no effect. Similar results were observed in mouse uterine strips. Following in vivo administration to pregnant mice, oxytocin receptor–targeted liposomes localized specifically to the uterine horns and mammary tissue. Targeting increased localization to the uterus 7-fold. Localization was not detected in the maternal brain or fetus. Targeted and nontargeted liposomes also localized to the liver. Oxytocin receptor–targeted liposomes loaded with indomethacin were effective in reducing rates of preterm birth in mice, whereas nontargeted liposomes loaded with indomethacin had no effect.
Conclusion
Our results demonstrate that oxytocin receptor–targeted liposomes can be used to either inhibit or enhance human uterine contractions in vitro. In vivo, the liposomes localized to the uterine tissue of pregnant mice and were effective in delivering agents for the prevention of inflammation-induced preterm labor. The potential clinical advantage of targeted liposomal drug delivery to the myometrium is reduced dose and reduced toxicity to both mother and fetus.
Click Supplemental Materials under article title in Contents at ajog.org
Introduction
Complications arising from preterm birth (PTB) are the leading cause of death among children age <5 years, accounting for nearly 1 million deaths in 2013,
Given that both can arise from dysregulation of uterine contractility, the need exists for safe and effective clinical interventions capable of modifying myometrial contractions to improve treatment of women in preterm labor, to induce or facilitate labor and to prevent or treat PPH, without adverse off-target effects on either the mother or fetus.
When a woman presents with preterm labor, attempts are often made to halt contractions by administering tocolytics that inhibit or block components of the contraction cascade. A recent study proposed that “the ideal tocolytic agent should be myometrium-specific, easy to administer, inexpensive, effective in preventing PTB and improve neonatal outcomes, with few maternal, fetal, and neonatal side effects, and without long-term adverse effects.”
Standard therapy varies from country to country, but tocolysis may involve the administration of calcium channel blockers, such as nifedipine (NIF); β2-adrenergic receptor agonists, such as salbutamol (SAL); an oxytocin receptor (OTR) antagonist, such as atosiban; or a prostaglandin synthetase inhibitor, such as indomethacin (IND).
Unfortunately, the systemic administration of these therapies and lack of specificity means that large doses need to be administered to achieve a therapeutic effect at the target tissue, the myometrium. Maternal side effects of β2-adrenergic receptor agonists include tremors, heart palpitations, and tachycardia, as well as myocardial ischemia and pulmonary edema.
NIF has been associated with fewer side effects, however approximately 1% of women experience a severe side effect and a further 1% experience mild adverse side effects.
An oxytocin receptor antagonist (atosiban) in the treatment of preterm labor: a randomized, double-blind, placebo-controlled trial with tocolytic rescue.
Achieving targeted drug delivery to the myometrium would reduce the quantity of drug required to achieve therapeutic efficacy, reduce the likelihood of maternal and fetal side effects, and would therefore represent a significant advancement for maternal-fetal medicine.
Targeted liposomes have emerged as a platform for achieving the delivery of drugs to specific tissues. Liposomes are artificial vesicles that range in size from 50-1000 nm, and are composed of ≥1 phospholipid bilayers.
Liposomal encapsulation improves the pharmacokinetics of drugs, particularly if the liposome surface is PEGylated, which reduces uptake by the reticuloendothelial system and prolongs half-life.
This has led to the development of liposomal-based preparations of various agents, including doxorubicin, amphotericin B, daunorubicin, and verteporfin.
Ligand-targeted liposomes offer the potential for site-specific delivery of drugs to designated cell types or organs in vivo that selectively express specific cell surface cognate receptors.
Although many types of targeting molecules are available, such as peptides/proteins and carbohydrates, the coupling of antibodies to the liposome surface to create immunoliposomes has many advantages. One advantage of using antibodies is their stability and higher binding avidity because of the presence of dual binding sites.
For example, liposomes coated with antibodies to intercellular adhesion molecule (ICAM)-1 have been developed for the treatment of inflammatory diseases.
Targeting of ICAM-1-directed immunoliposomes specifically to activated endothelial cells with low cellular uptake: use of an optimized procedure for the coupling of low concentrations of antibody to liposomes.
Administration of ICAM-1-targeted immunoliposomes loaded with an analgesic agent demonstrated specific localization and therapeutic efficacy exclusively in peripheral inflammatory tissue. All control groups (free drug solution, empty nontargeted liposomes, drug-loaded nontargeted liposomes, and empty ICAM-1-targeted immunoliposomes) showed no significant therapeutic response.
Targeted nanoparticles that mimic immune cells in pain control inducing analgesic and anti-inflammatory actions: a potential novel treatment of acute and chronic pain condition.
The aim of this study was to develop a means of targeting therapeutic agents to uterine myometrial tissue, to allow therapeutic modification of myometrial contractions in obstetric settings, such as preterm labor, labor induction, and PPH. The expression of the OTR is significantly up-regulated in myometrial cells approaching term.
Here we report the development of OTR-targeted PEGylated immunoliposomes loaded with traditional tocolytics, such as NIF and SAL, as well as rolipram (ROL), a phosphodiesterase (PDE)4 inhibitor and potent inhibitor of myometrial contractions.
Uterus-relaxing effect of beta2-agonists in combination with phosphodiesterase inhibitors: studies on pregnant rat in vivo and on pregnant human myometrium in vitro.
Moreover, we report enhancement of human myometrial contraction duration in vitro through liposomal delivery of dofetilide (DOF), a hERG channel blocker that increases myometrial contraction duration,
demonstrating that this delivery platform can be used to either inhibit or enhance contractions in human myometrial tissue. We demonstrate that intravenously (IV) administered OTR-targeted liposomes localize specifically to the uterine tissue of pregnant mice in vivo. Finally, using an inflammatory mouse model of PTB (lipopolysaccharide [LPS] administration), we show that OTR-targeted liposomes loaded with IND are effective in preventing PTB, while IND-loaded nontargeted liposomes have no effect.
Materials and Methods
Myometrial tissue acquisition
Human studies
These studies were performed in Newcastle, Australia, and were approved by the Hunter and New England Area Human Research Ethics Committee, adhering to guidelines of the University of Newcastle and John Hunter Hospital, Newcastle, Australia (02/06/12/3.13). All participants gave informed written consent. Collection of myometrial samples (5 × 5 × 10 mm) occurred from the lower uterine segment of term singleton pregnancies. All women were examined clinically, and those with signs of infection were excluded. Women were undergoing term elective cesarean delivery and were not in labor (NIL); the clinical indications for elective NIL cesarean delivery were previous cesarean delivery or previous third-/fourth-degree tear. All participants ranged from 37-40 completed weeks of gestation. Following delivery of the placenta, all women immediately received 5 U of oxytocin (Syntocinon) into an IV line, which was administered as standard care. Myometrial biopsies were excised 3-5 minutes after oxytocin administration, thus all samples were briefly exposed to oxytocin. After biopsy, myometrial samples were dissected from connective tissue and washed in ice-cold physiological saline.
Mouse in vitro studies
Mouse uterine horns were dissected from pregnant CD1 Swiss mice (8-10 weeks of age) at term gestation prior to the onset of labor (fetal gestation day [GA] 18). Mouse studies were approved by the University of Newcastle Animal Ethics Committee (A-2014-400/A-2014-429). All mice were housed under SPF/PC2 conditions under a 12-hour light-day cycle and had food and water available ad libitum.
Liposome manufacture
Liposomes containing NIF, SAL, ROL, DOF (each at approximately 4 mg/mL), or IND (approximately 5.5 mg/mL), as determined by high-performance liquid chromatography, were manufactured as previously outlined.
Targeting of ICAM-1-directed immunoliposomes specifically to activated endothelial cells with low cellular uptake: use of an optimized procedure for the coupling of low concentrations of antibody to liposomes.
Liposomes were composed of 1,2-distearoyl-sn-glycero-2-phosphocholine (DSPC) and cholesterol in a molar ratio of 2:1, and contained 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG(2000) maleimide) at 1.5 mol percent of DSPC as a coupling lipid (Avanti Polar Lipids). The resulting multilamellar dispersions were reduced in size and lamellarity to approximately 200 nm in diameter by high-pressure extrusion. The activated liposome suspension was then mixed with thiolated polyclonal anti-OTR antibody (catalog no. ab115664; Abcam, Cambridge, MA), which was prepared by first conjugating 25 μg of OTR antibody with a heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio) propionate (Figure 1). The OTR antibody recognizes an extracellular domain of the human OTR. Nontargeted liposomes were coated with rabbit IgG. All liposomes incorporated the membrane stain 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) for fluorescent detection. Unconjugated antibody and nonencapsulated drug was removed by centrifugal filtration of the liposomes through a 100-kd molecular weight filter (Amicon Ultra-15). Amicon Ultra-15 filters were washed with Milli-Q H2O before 500 μL of liposome suspension was loaded into the filter reservoir. Liposomes were diluted with 5 mL of sterile 0.9% saline and centrifuged at 4000 x g until retentate volume was <250 μL. Liposomes were then resuspended in a further 5 mL of 0.9% saline and centrifuged until retentate volume was <250 μL. Filtered liposomes were then collected, transferred to a fresh Eppendorf tube, and redispersed to an original volume of 500 μL.
Figure 1Schematic of oxytocin receptoretargeted liposome structure
The size distribution of the liposomal dispersion was determined by dynamic laser light scattering (Zetasizer Nano S, ATA Scientific). Encapsulation efficiency was determined by disrupting the vesicles with ethanol and evaluating drug concentration using high-performance liquid chromatography. Quantification of the amount of antibody associated with liposomes was determined using the CBQCA protein assay (ThermoFisher Scientific Inc, Waltham, MA), using bovine serum albumin for the preparation of the standard curve.
Myometrial contractility studies
Myometrial strips were set up as previously described.
Briefly, NIL human myometrial samples, or uterine horns obtained from pregnant CD1 Swiss mice, were dissected into strips (10 × 2 × 2 mm) and suspended in organ baths containing 30 mL of physiological saline solution (PSS) containing 120 mmol/L of NaCl, 5 mmol/L of KCl, 25 mmol/L of NaHCO3, 1 mmol/L of KH2PO4, 1.2 mmol/L of MgSO4, 2.5 mmol/L of CaCl2, and 11 mmol/L of glucose, and continuously gassed with carbogen (95% O2, 5% CO2) at pH 7.4. Strips were connected to a Grass FT03C force transducer (Grass Instruments, Quincy, MA) and 1g passive tension applied (1g was calibrated to equal 1 V). PSS was replaced 5 times during the first hour, with strips retensioned to 1g passive tension following each wash. Thereafter strips were maintained at 37°C until spontaneous rhythmic contractions developed. Data were digitized using a MacLab/8E data-acquisition system and contraction status visualized in real time using Chart software (ADInstruments, Dunedin, New Zealand). For each strip a contraction baseline was acquired to serve as reference.
To administer liposome treatments, 600 μL of PSS buffer was carefully extracted from an organ bath and transferred to an Eppendorf tube. The appropriate volume of liposome preparation (mixed by inversion) was pipetted into the PSS to predilute the liposomes. The total volume of prediluted liposomes (600 μL PSS + liposomes) was then carefully reinjected back into the appropriate organ bath. Final concentrations of each drug were: NIF 7.7 μmol/L, SAL 9.25 μmol/L, ROL 19.4 μmol/L, and DOF 3.0 μmol/L. Doses were based on prior investigations of the nonencapsulated drug (in vitro contraction assays using human myometrium). Where treated tissue was not washed, tissue strips remained in the presence of the liposomes for the duration of the assay. Where washout studies were performed, organ baths were twice drained of buffer and refilled with 37°C PSS. Human tissue strips were washed after 1 hour and 25 minutes whereas mouse tissue strips were washed after 15 minutes.
Tension generated by tissue strips is indicated in the results and representative contraction traces. The effect of treatments is interpreted relative to the pretreatment contraction baseline, which consisted of ≥3 contractions of consistent frequency and amplitude.
In vivo biodistribution study
Timed-mated CD1 Swiss pregnant mice were injected with drug-free, DiI-labeled preparations of either nontargeted or OTR-targeted liposomes on fetal GA 17 and 18 at 4:00 pm. Mice that labored overnight were euthanized on the morning of day 19 (9:00-11:00 am) by CO2 asphyxiation. Maternal internal organs of interest (heart, brain, liver, lung, kidney, uterus, and mammary tissue) were harvested and transferred to a Petri dish along with a sacrificed neonate. The Petri dish was loaded into an in vivo imaging system (IVIS-100) (Xenogen, Alameda, CA) and a light image captured. Tissues were then imaged under conditions appropriate for the detection of DiI (excitation: 554 nm; emission: 583 nm; filter: DsRed; exposure: 4 seconds; field of view: 10; binning: 4). Organs were imaged 17-19 hours after the second injection, following labor. Background signal was subtracted from the detected signal to produce the final fluorescence image. Fluorescence signal is reported as radiance (p/s/cm2/sr). The radiance range was kept constant across all images (min = 2.0 × 108; max = 1.8 × 109).
PTB study
Time-mated pregnant CD1 Swiss mice were administered 0.7 μg/g LPS from Escherichia coli (0111:B4) (Sigma-Aldrich) via intraperitoneal (IP) injection at 12:00 pm on GA 15 (1-time injection). LPS dose had been previously optimized to result in PTB rates of 50-70%. Total IP injection volume was 150 μL in saline.
At 4:00 pm on GA 15, mice began receiving daily IV injections of IND free-drug or liposomal preparations according to assigned treatment groups. Treatment groups are indicated in Table 1. Total IV injection volume was 150 μL. Mice were monitored for onset of labor every 6 hours. Treatments were repeated daily at 4:00 pm until all mice labored. Term gestation was 19-22 days. Mice that labored within 48 hours of receiving LPS (GA 17) were deemed to have labored preterm.
Table 1Treatment groups for in preterm labor study
Group
One-time IP injection (12:00 pm on GA 15, 150 μL)
Daily IV injections (4:00 pm, GA ≥15, 150 μL)
1
Saline
Saline
2
0.7 μg/g LPS
50% DMSO
3
0.7 μg/g LPS
1.0 mg/kg/d IND in 50% DMSO
4
0.7 μg/g LPS
2.0 mg/kg/d IND in 50% DMSO
5
0.7 μg/g LPS
OTR-targeted, drug-free liposomes in saline
6
0.7 μg/g LPS
2.0 mg/kg/d IND via nontargeted liposomes in saline
7
0.7 μg/g LPS
2.0 mg/kg/d IND via OTR-targeted liposomes in saline
For contraction traces, LabChart software (ADInstruments) was used to determine the area under the curve (AUC) (g tension × seconds) for the 30 minutes prior to treatment (pretreatment) and 30 minutes after treatment (posttreatment). AUC before and after treatment was compared by 2-tailed paired t test (GraphPad Prism).
For DOF studies, contraction plateau duration (seconds) was determined for 4 contractions pretreatment and posttreatment using LabChart software. Plateau duration was determined as the time between the point of highest amplitude and point where contraction force declined sharply. Contraction duration data were obtained for 3 individual tissues (n = 3 women). Pretreatment and posttreatment measurements (n = 12 each) were compared by 2-tailed unpaired t test.
Average radiance (p/s/cm2/sr) was determined for each organ of interest using Living Image software (v2.5). Where fluorescence was detected, regions of interest were applied automatically (contour). Where detection was low or absent, regions of interest were specified manually (circles or squares) to tightly encompass the tissue being analyzed. Data were tested for normality by the Shapiro-Wilk normality test (GraphPad Prism). Average radiance for each organ/tissue was compared between treatment groups (n = 4 animals per group) by 1-way analysis of variance (ANOVA) with multiple comparisons (Holm-Sidak) (GraphPad Prism).
For the preterm labor studies, rate of PTB was compared between treatment groups by χ2 analysis. Time (hours) between LPS injection and labor was calculated. Data were transformed (Y = Y2) to obtain normal distribution (D’Agostino and Pearson normality test) and analyzed by 1-way ANOVA with multiple comparisons (Tukey). Data recorded for number of pups for term deliveries were normally distributed (Shapiro-Wilk normality test) and analyzed by 1-way ANOVA with multiple comparisons (Tukey). Preterm deliveries did not yield any viable pups.
Consumables and reagents
Mice were supplied by the University of Newcastle Animal Support Unit. NIF (catalog no. 1075), SAL hemisulfate (catalog no. 0634), ROL (catalog no. 0905), and DOF (catalog no. 3757) were purchased from Tocris (Bristol, United Kingdom). IND (catalog no. L2630) was purchased from Sigma-Aldrich Pty Ltd (Sydney, Australia). Anti-OTR antibody (ab115664) was purchased from Abcam. Other miscellaneous reagents were purchased from Sigma-Aldrich Pty Ltd and ThermoFisher Scientific Inc.
Results
Characteristics of the liposomal delivery system
OTR-targeted PEGylated immunoliposomes had a mean particle size of 197 ± 6.8 nm with a polydispersity index of 0.243 ± 0.043 (mean ± SD; n = 3). The size and polydispersity of the control liposome formulations were similar. Encapsulation of therapeutic agents into the liposomes did not significantly affect the size or polydispersity index. Mean antibody coupling ratio for the OTR-targeted liposomes was 1.86 ± 0.17 μg of antibody/μmol of phospholipid. With a starting antibody concentration of 25 μg and a phospholipid concentration of 2.03 × 10–5 mol this equates to a conjugation efficiency of >99%. The liposomes have a neutral net charge and a drug loading efficiency of >95%, which equates to ∼4 mg/mL of drug encapsulated/mL of liposome suspension composed of 16 mg DSPC and 4 mg cholesterol (molar ratio 2:1). In vitro dialysis studies have demonstrated highly stable vesicles upon dilution in an aqueous phase (PBS pH 7.4) and in serum (50% FCS) at 37°C (data not included).
Human myometrial contractility
Contraction bioassays were performed to assess whether targeted liposomes were capable of delivering encapsulated therapeutic agents to modulate spontaneous human uterine contractions in vitro. Treating the uterine strips with OTR-targeted liposomes that contained no therapeutic agent (n = 3) (Figure 2, Ai) had no effect on myometrial contractility in that AUC was not affected (P = .08; pretreatment = 1790.0 ± 19.5; posttreatment = 1704.0 ± 38.8 g/s) (Figure 2, Aii). For each therapeutic agent examined in vitro, we prepared nontargeted (IgG-coated) and OTR-targeted (anti-OTR-coated) liposomes (at 4 mg/mL). Administering 7.7 μmol/L NIF to human uterine strips via nontargeted NIF-loaded liposomes (Figure 2, Bi) (n = 5) had no effect on contractility in that AUC was not affected (P = .4136; pretreatment = 1701.4 ± 55.9; posttreatment = 1643.8 ± 19.6 g/s) (Figure 2, Bii). Administering 7.7 μmol/L NIF via OTR-targeted NIF-loaded liposomes (Figure 2, Biii) (n = 4) resulted in abolition of myometrial contractions and a significant reduction in AUC (P = .0277; pretreatment = 1767.8 ± 15.5; posttreatment = 1137.5 ± 24.8 g/s) (Figure 2, Biv).
Figure 2Use of targeted liposomes to inhibit human uterine contractility in vitro
Data are contraction traces for strips of human myometrial tissue and corresponding area under the curve (AUC) analyses. A, Effect of oxytocin receptor (OTR)-targeted, drug-free control liposomes on myometrial contractions in vitro (n = 3). B, Effect of nontargeted (n = 5) and OTR-targeted (n = 4) nifedipine (NIF)-loaded liposomes on myometrial contractions in vitro. C, Effect of nontargeted (n = 3) and OTR-targeted (n = 3) salbutamol (SAL)-loaded liposomes on myometrial contractions in vitro. Average AUC analyses covers 30 minutes immediately prior to and 30 minutes after treatment with NIF- or SAL-loaded liposomes (pretreatment and posttreatment, respectively). AUC analyses are paired t tests.
Paul et al. Targeted uterine drug delivery system. Am J Obstet Gynecol 2017.
Administering 9.25 μmol/L SAL to spontaneously contracting human uterine strips via nontargeted SAL-loaded liposomes (Figure 2, Ci) (n = 3) had no effect on contractility with AUC being unaffected by the treatment (P = .2022; pretreatment = 1775.3 ± 24.8; posttreatment = 1749.3 ± 16.4 g/s) (Figure 2, Cii). Administering 9.25 μmol/L SAL via OTR-targeted SAL-loaded liposomes (Figure 2, Ciii) (n = 3) resulted in complete abolition of contractions and significant reduction in AUC (P = .0293; pretreatment = 1749.7 ± 27.3; posttreatment = 1292.0 ± 77.1 g/s) (Figure 2, Civ). Similar results were observed when ROL was encapsulated in nontargeted and OTR-targeted liposomes (Figure 4).
To demonstrate that our OTR-targeted liposomes are capable of functioning as a drug delivery system for different obstetric applications, such as treating PPH, liposomes were prepared that contained the hERG channel blocker, DOF. When administered to human myometrial tissue, DOF increases the contraction duration and reduces contraction frequency by delaying repolarization of the myocyte membrane.
These results demonstrate that a single delivery system, OTR-targeted liposomes, can be utilized to deliver either contraction-blocking or contraction-promoting therapeutics to uterine myocytes.
Figure 3Use of targeted liposomes to enhance human myometrial contractions in vitro
Data are contraction trace analyses for strips of human myometrial tissue. A, Effect of 3.0 μmol/L dofetilide (DOF) administered via nontargeted (n = 3) or oxytocin receptor (OTR)-targeted (n = 3) DOF-loaded liposomes on contractility in vitro. B, Average contraction plateau duration for 4 contractions immediately prior to and after treatment with nontargeted or OTR-targeted DOF-loaded liposomes (pretreatment and posttreatment, respectively) (n = 3 tissues strips each). Unpaired t test (12 pretreatment plateau durations vs 12 posttreatment plateau durations).
Paul et al. Targeted uterine drug delivery system. Am J Obstet Gynecol 2017.
Data are contraction traces for individual strips of human myometrial tissue. A, Effect of nontargeted (n = 3) and oxytocin receptor (OTR)-targeted (n = 3) rolipram (ROL)-loaded liposomes on myometrial contractions in vitro. Aii, Restoration of contractions after washout. B, Average area under the curve (AUC) for 30 minutes immediately prior to and 30 minutes after treatment with ROL-loaded liposomes (pretreatment and posttreatment, respectively). AUC analyses were paired t tests.
Paul et al. Targeted uterine drug delivery system. Am J Obstet Gynecol 2017.
The effect of nontargeted and OTR-targeted DOF-loaded liposomes on AUC was analyzed. Neither treatment significantly affected AUC (data not shown).
Effect of targeted liposomes is reversible
To demonstrate that the effects on contractility were due to pharmacological actions of the drugs and not the result of toxic effects of OTR-targeted liposomes, washout experiments were performed. ROL is a reversible inhibitor of PDE4 that induces myometrial relaxation.
Correlation between selective inhibition of the cyclic nucleotide phosphodiesterases and the contractile activity in human pregnant myometrium near term.
Administering 19.4 μmol/L ROL to contracting strips via nontargeted ROL-loaded liposomes (Figure 4, Ai) (n = 3) had no effect. When 19.4 μmol/L ROL was administered via OTR-targeted ROL-loaded liposomes (Figure 4, Aii) (n = 3), contractions were abolished. Analyses of contraction data indicated no reduction in AUC following treatment with nontargeted ROL-loaded liposomes (P = .061; pretreatment = 1657.0 ± 21.0; posttreatment = 1611.7 ± 14.9 g/s) (Figure 4, Bi), whereas AUC was significantly reduced following treatment with 19.4 μmol/L ROL administered via OTR-targeted ROL-loaded liposomes (P = .0023; pretreatment = 1648.1 ± 14.3; posttreatment = 1155.7 ± 36.3 g/s) (Figure 4, Bii). Once contractions were inhibited for 1 hour and 25 minutes, tissue strips were washed twice in PSS and monitored. Spontaneous, rhythmic contractions resumed in myometrial strips previously treated with OTR-targeted ROL-loaded liposomes (Figure 4, Aii), indicating that the tissue remained viable.
Mouse myometrial contractility
Prior to commencing mouse in vivo studies, we confirmed that liposomes were effective in delivering therapeutic agents to mouse uterine tissue in vitro. Results observed in the mouse were consistent with human myometrial contractility studies. OTR-targeted, drug-free liposomes (a control preparation) had no effect on mouse uterine contractions (Figure 5, A) (n = 3). Administering 9.25 μmol/L SAL via nontargeted (IgG-coated control) SAL-loaded liposomes had no effect on contractility (Figure 5, Bi) (n = 3), whereas the same SAL dose administered via OTR-targeted SAL-loaded liposomes abolished mouse myometrial contractions in vitro (Figure 5, Bii) (n = 3). Spontaneous contractions resumed following washing of tissue strips, demonstrating that the mouse uterine tissue remained viable following administration of the liposomes. Similar results were obtained for liposomes loaded with NIF (data not shown). These results demonstrated that OTR-targeted liposomes were effective in modulating mouse myometrial contractility.
Figure 5Use of targeted liposomes to modulate mouse uterine contractility in vitro
Data are contraction traces for individual strips of mouse uterine tissue and illustrate effect of treatments on contraction amplitude and frequency. A, Effect of oxytocin receptor (OTR)-targeted, drug-free liposomes (n = 3). Bi, Effect of 9.25 μmol/L salbutamol (SAL) administered via nontargeted SAL-loaded liposomes (n = 3). Bii, Effect of 9.25 μmol/L SAL administered via OTR-targeted SAL-loaded liposomes (n = 3).
Paul et al. Targeted uterine drug delivery system. Am J Obstet Gynecol 2017.
We examined the biodistribution of DiI-labeled nontargeted (naked) and OTR-targeted liposomes that occurred in vivo. Pregnant mice were injected with liposomes approaching term (GA 17 and 18) then sacrificed shortly after labor (GA 19) (17-19 hours after injection). Whole organs (liver, brain, heart, kidney, lung, mammary tissue, and uterus) were placed on Petri dishes, along with a euthanized neonate, and imaged. The arrangement of tissues (Figure 6, A) was kept consistent when imaging tissues from different mice. DiI does not readily exchange out of liposomes into cell membranes or other lipid-containing structures, and therefore is an appropriate marker to assess the biodistribution of liposomes.
Figure 6Oxytocin receptor (OTR)-targeted liposomes accumulate in uterus in vivo
Data are light or fluorescence images captured shortly after labor by IVIS-100 (Xenogen, Alameda, CA) illustrating liposome biodistribution that occurred in vivo. A, Representative image demonstrating arrangement of organs and tissues of interest (liver, brain, lung, heart, kidney, uterus, mammary tissue, and neonate). B, Fluorescent detection of liposome biodistribution that occurred in vivo. Biodistribution of nontargeted liposomes (n = 4; animals 1–4) and OTR-targeted liposomes (n = 4; animals 5–8). C, Quantitation of liposomal detection in different organs. Average radiance (p/s/cm2/sr) was determined for each organ and compared across treatment groups (n = 4 for each organ per group). Data were confirmed to be normally distributed (Shapiro-Wilk normality test) then compared by 1-way analysis of variance with multiple comparisons (Holm-Sidak). Not all statically significant comparisons are indicated.
Paul et al. Targeted uterine drug delivery system. Am J Obstet Gynecol 2017.
Fluorescence detection (p/s/cm2/sr) of nontargeted liposomes injected into pregnant mice consistently revealed liposome accumulation in the liver, which is the site of liposome clearance from the blood stream and metabolism.
Accumulation of nontargeted liposomes was not detected in the brain, heart, kidney, lung, mammary tissue, or uterus nor in the neonates (n = 4 each) (Figure 6B; animals 1 - 4). Organs isolated from mice injected with OTR-targeted liposomes showed accumulation of the OTR-targeted liposomes in the uterus and the mammary glands. As expected, there were also high levels of liposome localization in the liver. OTR-targeted liposome accumulation was not detected in the brain, heart, kidney, or lung, nor in the neonates (n = 4 each) (Figure 6B; animals 5 - 8).
DiI fluorescence was quantified for each organ (Table 2). OTR targeting of liposomes resulted in significantly increased localization to the uterus compared to nontargeted liposomes (P < .0001; nontargeted = 5.04 × 107 ± 7.5 × 106; OTR-targeted = 3.57 × 108 ± 3.05 × 107 p/s/cm2/sr) (Figure 6, C). On average this equaled a 7-fold increase in uterine localization. Furthermore, the level of OTR-targeted liposome localization in the uterus was significantly greater than that of brain (P = .0002), lung (P = .0003), kidney (P = .0005), heart (P < .0001), and neonate (P < .0001) (Figure 6, C). For both nontargeted and OTR-targeted liposomes, accumulation in the liver was significantly greater than all other organs examined (P < .0001), however there was no difference in liposome accumulation in the liver between nontargeted and OTR-targeted liposomes (P > .9999; nontargeted = 7.90 × 108 ± 9.15 × 107; OTR-targeted = 7.37 × 108 ± 9.05 × 107) (Figure 6, C).
Table 2Average radiance of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate–labeled liposomes detected in organs and neonates
Organ/tissue
Average radiance, p/s/cm2/sr Mean ± SEM
Nontargeted liposomes n = 4 animals
OTR-targeted liposomes n = 4 animals
Liver
9.73 × 108 ± 9.1 × 107
7.37 × 108 ± 9.05 × 107
Uterus
5.04 × 107 ± 7.5 × 106
3.57 × 108 ± 3.05 × 107
Mammary tissue
7.29 × 107 ± 6.05 × 106
1.78 × 108 ± 6.47 × 107
Brain
3.97 × 107 ± 2.51 × 106
7.03 × 107 ± 4.51 × 106
Lung
4.65 × 107 ± 4.82 × 106
7.97 × 107 ± 2.94 × 106
Kidney
3.46 × 107 ± 1.78 × 106
8.79 × 107 ± 9.51 × 106
Heart
2.79 × 107 ± 1.65 × 106
5.52 × 107 ± 1.65 × 106
Neonate
–4.83 × 107 ± 1.30 × 107
–1.03 × 107 ± 1.46 × 107
OTR, oxytocin receptor.
Paul et al. Targeted uterine drug delivery system. Am J Obstet Gynecol 2017.
We used an LPS model of PTB to assess whether targeted liposomes could be used to administer IND for the prevention of LPS-induced PTB in mice. Nontargeted and OTR-targeted liposomes loaded with 5.5 mg/mL IND were compared against IND administered as free drug (1.0 or 2.0 mg/kg/d). Observed PTB rates are indicated in Table 3. The χ2 analyses were performed.
Table 3Rates of lipopolysaccharide-induced preterm birth and time between lipopolysaccharide injection and labor
Group no.
Treatment group
n
PTB rate (%)
Time between LPS injection and observed labor, h, mean ± SEM
PTB rates in control mice (group 1) and the LPS control group (group 2) were 0 (n = 12) and 67% (n = 18), respectively. At 2.0 mg/kg/d, IND administered as free drug significantly reduced rates of PTB from 67% down to 31% (group 2 [n = 18] vs group 4 [n = 13]; P = .0484) (Figure 7, A). PTB rate for OTR-targeted, drug-free control liposomes (group 5) was 56% (n = 16), and was not different to PTB rate observed for LPS control animals (group 2) (P = .532). IND administered at 2.0 mg/kg/d via nontargeted liposomes (group 6) (n = 12) had no effect as the observed PTB rate of 58% was not significantly different from PTB rates for LPS control animals (group 2) (P = .643) or animals treated with OTR-targeted, drug-free liposomes (group 5) (P = .91) (Figure 7, A).
Efficacy of targeted liposomes was assessed using lipopolysaccharide (LPS) mouse model of PTB. A, Effect of IND administered as free drug (1.0 or 2.0 mg/kg/d) or via liposomal preparations (2.0 mg/kg/d) on rates of LPS-induced PTB. B, Time (hours) between LPS injection and observed labor. C, Number of live pups born for term deliveries. No significant differences were recorded in number of live pups. PTB rates were analyzed by χ2 analysis. Data for time (hours) between LPS injection and labor were normalized (Y = Y2) (D’Agostino and Pearson normality test) then analyzed by 1-way analysis of variance (ANOVA) with multiple comparisons (Tukey). Number of live pups was normally distributed and analyzed by 1-way ANOVA with multiple comparisons (Tukey).
DMSO, dimethyl sulfoxide.
Paul et al. Targeted uterine drug delivery system. Am J Obstet Gynecol 2017.
IND administered at 2.0 mg/kg/d via OTR-targeted liposomes (group 7) (n = 11) resulted in a PTB rate of 18%, which was a significant reduction compared to the PTB rate of 67% for the LPS control animals (group 2) (P = .0112) (Figure 7, A). Furthermore, PTB rate for 2.0 mg/kg/d IND administered via OTR-targeted liposomes was significantly reduced compared to the same dose administered by nontargeted liposomes (group 6) (P = .048). No significant difference was observed between 2.0 mg/kg/d IND administered as free drug compared to when administered via OTR-targeted liposomes (group 4 vs group 7; P = .4780) (Figure 7, A).
The time between LPS injection and labor was calculated for each animal (average ± SEM shown in Table 3). Analysis of the normalized data showed that IP administration of LPS (0.7 μg/g) significantly advanced the time of labor, compared to control animals (group 1 vs group 2; P = .0017) (Figure 7, B). IND administered as free drug at 1.0 and 2.0 mg/kg/d dose-dependently increased the average time between LPS injection and labor (77.5 ± 14.1 and 86.6 ± 12.7 hours, respectively) compared to the LPS control (50.8 ± 8.9 hours), however neither dose reached statistical significance (group 2 vs group 3; P = .53, and group 2 vs group 4; P = .08) (Figure 7, B). OTR-targeted, drug-free liposomes had no effect on time between LPS injection and labor, compared to LPS control animals (group 2 vs group 5; P = .92). A total of 2.0 mg/kg/d IND administered via nontargeted liposomes had no significant effect on the time between LPS injection and labor (group 2 vs group 6; P = .99), however, when administered via OTR-targeted liposomes, time between LPS injection and labor was significantly increased (group 2 vs group 7; P = .0048). The time between LPS injection and labor was significantly different between 2.0 mg/kg/d IND delivered via nontargeted liposomes compared to OTR-targeted liposomes (group 6 vs group 7; P = .0438).
Number of live pups was recorded for term deliveries (no viable pups arose from preterm deliveries). Data were normally distributed (Shapiro-Wilk normality test) and analyzed by 1-way ANOVA with multiple comparisons (Tukey). There was no significant difference in the number of live pups from term deliveries in the different groups (Figure 7, C).
Comment
Principal findings
OTRs are expressed at low levels on various tissues toward the end of pregnancy, including the brain and mammary tissue.
indicating that the OTR is an excellent candidate for the development of a targeted drug delivery system for the uterus. This study represents an initial analysis of OTR-targeted liposomes as a drug delivery system, and demonstrates that:
(i)
conjugation of the OTR antibody to the surface of liposomes confers the ability for NIF-, SAL-, and ROL-loaded liposomes to significantly reduce human myometrial contractions in vitro, as confirmed by AUC analyses;
(ii)
enhancement of myometrial contractility can be achieved through encapsulation of uterotonic agents, as confirmed by use of OTR-targeted DOF-loaded liposomes to significantly increase contraction plateau duration;
(iii)
nontargeted liposomes loaded with these same therapeutic agents do not affect myometrial contractions in vitro, as confirmed by AUC and contraction plateau duration analyses;
(iv)
the effects are reversible (depending on the therapeutic), as confirmed by the spontaneous resumption of contractions in both human and mouse myometrial tissue in vitro;
(v)
the OTR-targeted liposomes themselves have no apparent effect on myometrial contractions, as confirmed by AUC analyses for myometrial contractions in vitro, and lack of effect on PTB rates in mice or time between LPS injection and labor;
(vi)
in vivo, OTR-targeted liposomes localize to the uterus and breast of pregnant mice whereas nontargeted liposomes do not. Uterine localization was increased 7-fold by OTR targeting, as confirmed by quantitation of average radiance for key organs of interest;
(vii)
no evidence of transplacental passage of the liposomes to the fetus was observed, as determined quantitative evaluation of DiI fluorescence in neonates;
(viii)
OTR-targeted liposomes loaded with IND are effective in reducing rates of LPS-induced PTB in mice whereas nontargeted IND-loaded liposomes have no effect.
Clinical significance
Many current tocolytics have been associated with adverse effects on the mother (β-sympathomimetics)
Safety concerns for the use of calcium channel blockers in pregnancy for the treatment of spontaneous preterm labor and hypertension: a systematic review and meta-regression analysis.
An oxytocin receptor antagonist (atosiban) in the treatment of preterm labor: a randomized, double-blind, placebo-controlled trial with tocolytic rescue.
NIF is capable of providing some clinical benefit, with a systematic review and meta-analysis indicating a significant reduction in the risk of delivery within 7 days of initiation of NIF treatment.
Safety concerns for the use of calcium channel blockers in pregnancy for the treatment of spontaneous preterm labor and hypertension: a systematic review and meta-regression analysis.
A double-blind randomized study of fetal side effects during and after the short-term maternal administration of indomethacin, sulindac, and nimesulide for the treatment of preterm labor.
however, systematic review indicates that IND is associated with an increased risk for severe intraventricular hemorrhage, necrotizing enterocolitis, and periventricular leukomalacia.
Antenatal exposure to indomethacin increases the risk of severe intraventricular hemorrhage, necrotizing enterocolitis, and periventricular leukomalacia: a systematic review with metaanalysis.
demonstrated that encapsulation of IND inside nontargeted liposomes can reduce IND levels in the fetus 7.6-fold, suggesting the potential for reduced fetal side effects.
Here we have demonstrated in mice that IND encapsulated inside OTR-targeted liposomes was effective in reducing rates of LPS-induced PTB, whereas nontargeted IND-loaded liposomes were not. These results, in conjunction with our biodistribution studies, suggest that OTR targeting confers upon liposomes the ability to target therapeutics to the uterus. The clinical implications are that OTR-targeted liposomes may enable existing tocolytics, such as NIF and IND, to be administered with improved efficacy and improved safety.
The restricted biodistribution of OTR-targeted liposomes raises the possibility of introducing into clinical practice therapeutic agents that are known to be highly effective tocolytics, yet are known to have adverse off-target effects. One such group of candidates are the PDE4 inhibitors, such as ROL, which have been demonstrated to be highly effective in controlling inflammation-driven preterm delivery in mice.
Mounting evidence indicates that PTB in human beings is also an inflammation-driven event, and evidence that ROL is highly effective in abolishing spontaneous contractions in human myometrium
suggests that PDE4 inhibitors may be excellent cargo for OTR-targeted liposomes in the setting of preterm labor.
Clinical implications also include the prospect of encapsulating uterotonic agents, and here we demonstrate that possibility through the use of DOF. DOF is not a traditional uterotonic agent, but when administered via OTR-targeted liposomes DOF significantly increased the duration of human myometrial contractions in vitro, consistent with previous findings.
Targeted delivery of uterotonics may be useful to promote contractions, including during failure of labor to progress, expulsion of the placenta after labor, expulsion of retained products after miscarriage, or to control PPH. PPH is a leading cause of maternal mortality worldwide and is linked with major morbidities such as peripartum hysterectomy and massive transfusion.
First-line therapy for PPH is uterine massage and oxytocin administration, however rates of atonic PPH after oxytocin use are increasing in many developed countries.
When refractory uterine atony occurs, second-line therapy may include administration of uterotonic agents such as methylergonovine and carboprost. Methylergonovine was recently identified as the more effective of the two,
however both agents could effectively be encapsulated in OTR-targeted liposomes. Evidence indicates that postreceptor contractile signaling pathways are maintained in oxytocin desensitized primary myocytes in vitro,
Uterotonic-loaded liposomes targeted to the OTR may therefore be of reduced effectiveness in patients with prolonged exposure to oxytocin.
Future research
These data provide the first evidence that OTR-targeted liposomes are a drug delivery system that affords flexibility in delivery of different classes of therapeutic agents to human uterine tissue to modulate myometrial contractility. Furthermore, these data provide the first evidence that OTR-targeted liposomes can be used to administer therapeutic agents for the prevention of PTB in mice.
Further studies are necessary to determine the mechanism of OTR-targeted liposome uptake in myocytes, the quantitative biodistribution of therapeutic agents achieved in the uterus compared to other organs, and the rate of liposome clearance. Additional studies have been planned to determine whether the use of OTR-targeted liposomes to administer therapeutics for prevention of PTB is effective in reducing fetal side effects, such as premature closure of the ductus arteriosus in response to IND exposure in utero.
Acknowledgment
The authors wish to thank the obstetricians from the John Hunter Hospital; our research midwife, Anne Wright; and research participants who donated samples toward this study. We thank Dr Gough Au, Dr Min Yuan Quah, Dr Yvonne Wong, and Jack Hockley for assistance with the in vivo imaging system, in particular Dr Gough Au for assistance with in vivo imaging system quantitation. We thank Professor Tamas Zakar for assistance with the statistical analyses of all data.
An oxytocin receptor antagonist (atosiban) in the treatment of preterm labor: a randomized, double-blind, placebo-controlled trial with tocolytic rescue.
Targeting of ICAM-1-directed immunoliposomes specifically to activated endothelial cells with low cellular uptake: use of an optimized procedure for the coupling of low concentrations of antibody to liposomes.
Targeted nanoparticles that mimic immune cells in pain control inducing analgesic and anti-inflammatory actions: a potential novel treatment of acute and chronic pain condition.
Uterus-relaxing effect of beta2-agonists in combination with phosphodiesterase inhibitors: studies on pregnant rat in vivo and on pregnant human myometrium in vitro.
Correlation between selective inhibition of the cyclic nucleotide phosphodiesterases and the contractile activity in human pregnant myometrium near term.
Safety concerns for the use of calcium channel blockers in pregnancy for the treatment of spontaneous preterm labor and hypertension: a systematic review and meta-regression analysis.
A double-blind randomized study of fetal side effects during and after the short-term maternal administration of indomethacin, sulindac, and nimesulide for the treatment of preterm labor.
Antenatal exposure to indomethacin increases the risk of severe intraventricular hemorrhage, necrotizing enterocolitis, and periventricular leukomalacia: a systematic review with metaanalysis.
This work was supported by grants from the National Health and Medical Research Council (Drs Hua and Smith), Hunter Medical Research Institute (Drs Paul, Hua, and Smith), and Global Alliance to Prevent Prematurity and Stillbirth. The funding providers had no involvement in the study or production of this article.
Drs Paul, Hua, and Smith hold a patent at the University of Newcastle related to the use of targeted liposomes. The other authors have no conflicts of interest to disclose.
Cite this article as: Paul JW, Hua S, Ilicic M, et al. Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor. Am J Obstet Gynecol 2017;216:283.e1-14.
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