Placental Drug Delivery to Treat Pre-Eclampsia and Fetal Growth Restriction.

Pre-eclampsia and fetal growth restriction (FGR) continue to cause unacceptably high levels of morbidity and mortality, despite significant pharmaceutical and technological advances in other disease areas. The recent pandemic has also impacted obstetric care, as COVID-19 infection increases the risk of poor pregnancy outcomes. This review explores the reasons why it lacks effective drug treatments for the placental dysfunction that underlies many common obstetric conditions and describes how nanomedicines and targeted drug delivery approaches may provide the solution to the current drug drought. The ever-increasing range of biocompatible nanoparticle formulations available is now making it possible to selectively deliver drugs to uterine and placental tissues and dramatically limit fetal drug transfer. Formulations that are refractory to placental uptake offer the possibility of retaining drugs within the maternal circulation, allowing pregnant individuals to take medicines previously considered too harmful to the developing baby. Liposomes, ionizable lipid nanoparticles, polymeric nanoparticles, and adenoviral vectors have all been used to create efficacious drug delivery systems for use in pregnancy, although each approach offers distinct advantages and limitations. It is imperative that recent advances continue to be built upon and that there is an overdue investment of intellectual and financial capital in this field.

Translational use of homing peptides: Tumor and placental targeting.

HYPOTHESIS: Tissue-specific homing peptides have been shown to improve chemotherapeutic efficacy due to their trophism for tumor cells. Other sequences that selectively home to the placenta are providing new and safer therapeutics to treat complications in pregnancy. Our hypothesis is that the placental homing peptide RSGVAKS (RSG) may have binding affinity to cancer cells, and that insight can be gained into the binding mechanisms of RSG and the tumor homing peptide CGKRK to model membranes that mimic the primary lipid compositions of the respective cells. EXPERIMENTS: Following cell culture studies on the binding efficacy of the peptides on a breast cancer cell line, a systematic translational characterization is delivered using ellipsometry, Brewster angle microscopy and neutron reflectometry of the extents, structures, and dynamics of the interactions of the peptides with the model membranes on a Langmuir trough. FINDINGS: We start by revealing that RSG does indeed have binding affinity to breast cancer cells. The peptide is then shown to exhibit stronger interactions and greater penetration than CGKRK into both model membranes, combined with greater disruption to the lipid component. RSG also forms aggregates bound to the model membranes, yet both peptides bind to a greater extent to the placental than cancer model membranes. The results demonstrate the potential for varying local reservoirs of peptide within cell membranes that may influence receptor binding. The innovative nature of our findings motivates the urgent need for more studies involving multifaceted experimental platforms to explore the use of specific peptide sequences to home to different cellular targets.

Liposome-Encapsulated Anti-inflammatory Proteins for Targeted Delivery to the Placenta to Treat Fetal Growth Restriction.

Fetal growth restriction (FGR), the failure of a fetus to reach its genetically determined growth potential, is a serious complication affecting up to 10% of pregnancies. FGR is a major risk factor for stillbirth and, in the survivors, neurodevelopmental disorders. We have recently identified that the anti-inflammatory and pro-resolving molecule, lipoxin A4 (LXA4) and its soluble receptor, formyl-peptide receptor-2 (FPR-2) are significantly decreased in human placentas from FGR pregnancy. The LXA4 synthetic analog Compound 43 (C43) is considered a safe, anti-inflammatory therapy and is being developed as a treatment for disease conditions with an inflammatory basis, for example, asthma in children. Identification of therapies to treat FGR in utero comes with the need to mitigate their potential side effects and the use of nanoparticle-mediated delivery systems could facilitate this. Our current studies are focused on targeting the resolution of inflammation observed in FGR placentas, by synthesizing liposome-encapsulated C43 as a novel therapeutic to improve placental function in FGR. In this chapter, we provide a detailed methodological procedure for the preparation of liposomes and conjugation of the peptide sequences, which selectively bind to the outer placental syncytiotrophoblast layer or the vascular endothelium of the uterine spiral arterioles.

Acute respiratory distress syndrome: potential of therapeutic interventions effective in treating progression from COVID-19 to treat progression from other illnesses-a systematic review.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is the most severe form of lung injury, rendering gaseous exchange insufficient, leading to respiratory failure. Despite over 50 years of research on the treatment of ARDS when developed from illnesses such as sepsis and pneumonia, mortality remains high, and no robust pharmacological treatments exist. The progression of SARS-CoV-2 infections to ARDS during the recent global pandemic led to a surge in the number of clinical trials on the condition. Understandably, this explosion in new research focused on COVID-19 ARDS (CARDS) rather than ARDS when developed from other illnesses, yet differences in pathology between the two conditions mean that optimal treatment for them may be distinct. AIM: The aim of the present work is to assess whether new therapeutic interventions that have been developed for the treatment of CARDS may also hold strong potential in the treatment of ARDS when developed from other illnesses. The study objectives are achieved through a systematic review of clinical trials. RESULTS: The COVID-19 pandemic led to the identification of various therapeutic interventions for CARDS, some but not all of which are optimal for the management of ARDS. Interventions more suited to CARDS pathology include antithrombotics and biologic agents, such as cytokine inhibitors. Cell-based therapies, on the other hand, show promise in the treatment of both conditions, attributed to their broad mechanisms of action and the overlap in the clinical manifestations of the conditions. A shift towards personalised treatments for both CARDS and ARDS, as reflected through the increasing use of biologics, is also evident. CONCLUSIONS: As ongoing CARDS clinical trials progress, their findings are likely to have important implications that alter the management of ARDS in patients that develop the condition from illnesses other than COVID-19 in the future.

Internal Mammary Arteries as a Model to Demonstrate Restoration of the Impaired Vasodilation in Hypertension, Using Liposomal Delivery of the CYP1B1 Inhibitor, 2,3',4,5'-Tetramethoxystilbene.

A significant number of patients with severe cardiovascular disease, undergoing coronary artery bypass grafting (CABG), present with hypertension. While internal mammary arteries (IMAs) may be a better alternative to vein grafts, their impaired vasodilator function affects their patency. Our objectives were to (1) determine if inhibition of the cytochrome P450 enzyme CYP1B1, using liposome-encapsulated 2,3',4,5'-tetramethoxystilbene (TMS), can potentiate vasodilation of IMAs from CABG patients, and (2) assess mechanisms involved using coronary arteries from normal rats, in an ex vivo model of hypertension. PEGylated liposomes were synthesized and loaded with TMS (mean diameter 141 ± 0.9 nm). Liposomal delivery of TMS improved its bioavailability Compared to TMS solution (0.129 ± 0.02 ng/mL vs. 0.086 ± 0.01 ng/mL at 4 h; p < 0.05). TMS-loaded liposomes alleviated attenuated endothelial-dependent acetylcholine (ACh)-induced dilation in diseased IMAs (@ACh 10−4 M: 56.9 ± 5.1%; n = 8 vs. 12.7 ± 7.8%; n = 6; p < 0.01) for TMS-loaded liposomes vs. blank liposomes, respectively. The alleviation in dilation may be due to the potent inhibition of CYP1B1 by TMS, and subsequent reduction in reactive oxygen species (ROS) moieties and stimulation of nitric oxide synthesis. In isolated rat coronary arteries exposed to a hypertensive environment, TMS-loaded liposomes potentiated nitric oxide and endothelium-derived hyperpolarization pathways via AMPK. Our findings are promising for the future development of TMS-loaded liposomes as a promising therapeutic strategy to enhance TMS bioavailability and potentiate vasodilator function in hypertension, with relevance for early and long-term treatment of CABG patients, via the sustained and localized TMS release within IMAs.

Placental expression of estrogen-related receptor gamma is reduced in fetal growth restriction pregnancies and is mediated by hypoxia†.

Fetal growth restriction (FGR) describes a fetus which has not achieved its genetic growth potential; it is closely linked to placental dysfunction and uteroplacental hypoxia. Estrogen-related receptor gamma (ESRRG) is regulated by hypoxia and is highly expressed in the placenta. We hypothesized ESRRG is a regulator of hypoxia-mediated placental dysfunction in FGR pregnancies. Placentas were collected from women delivering appropriate for gestational age (AGA; n = 14) or FGR (n = 14) infants. Placental explants (n = 15) from uncomplicated pregnancies were cultured for up to 4 days in 21% or 1% O2, or with 200 µM cobalt chloride (CoCl2), or treated with the ESRRG agonists DY131 under different oxygen concentrations. RT-PCR, Western blotting, and immunochemistry were used to assess mRNA and protein levels of ESRRG and its localization in placental tissue from FGR or AGA pregnancies, and in cultured placental explants. ESRRG mRNA and protein expression were significantly reduced in FGR placentas, as was mRNA expression of the downstream targets of ESRRG, hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2), and cytochrome P-450 (CYP19A1.1). Hypoxia-inducible factor 1-alpha protein localized to the nuclei of the cytotrophoblasts and stromal cells in the explants exposed to CoCl2 or 1% O2. Both hypoxia and CoCl2 treatment decreased ESRRG and its downstream genes' mRNA expression, but not ESRRG protein expression. DY131 increased the expression of ESRRG signaling pathways and prevented abnormal cell turnover induced by hypoxia. These data show that placental ESRRG is hypoxia-sensitive and altered ESRRG-mediated signaling may contribute to hypoxia-induced placental dysfunction in FGR. Furthermore, DY131 could be used as a novel therapeutic approach for the treatment of placental dysfunction.

Sex-specific effects of bisphenol A on the signaling pathway of ESRRG in the human placenta†.

Bisphenol A (BPA) exposure during pregnancy is associated with low fetal weight, particularly in male fetuses. The expression of estrogen-related receptor gamma (ESRRG), a receptor for BPA in the human placenta, is reduced in fetal growth restriction. This study sought to explore whether ESRRG signaling mediates BPA-induced placental dysfunction and determine whether changes in the ESRRG signaling pathway are sex-specific. Placental villous explants from 18 normal term pregnancies were cultured with a range of BPA concentrations (1 nM-1 µM). Baseline BPA concentrations in the placental tissue used for explant culture ranged from 0.04 to 5.1 nM (average 2.3 ±1.9 nM; n = 6). Expression of ESRRG signaling pathway constituents and cell turnover were quantified. BPA (1 µM) increased ESRRG mRNA expression after 24 h in both sexes. ESRRG mRNA and protein expression was increased in female placentas treated with 1 µM BPA for 24 h but was decreased in male placentas treated with 1 nM or 1 µM for 48 h. Levels of 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) and placenta specific-1 (PLAC1), genes downstream of ESRRG, were also affected. HSD17B1 mRNA expression was increased in female placentas by 1 µM BPA; however, 1 nM BPA reduced HSD17B1 and PLAC1 expression in male placentas at 48 h. BPA treatment did not affect rates of proliferation, apoptosis, or syncytiotrophoblast differentiation in cultured villous explants. This study has demonstrated that BPA affects the ESRRG signaling pathway in a sex-specific manner in human placentas and a possible biological mechanism to explain the differential effects of BPA exposure on male and female fetuses observed in epidemiological studies.

Conjugation to PEG as a Strategy to Limit the Uptake of Drugs by the Placenta: Potential Applications for Drug Administration in Pregnancy.

Here, we evaluated the feasibility of non-prodrug PEG-drug conjugates to decrease the accumulation of drugs within the placental tissues. The results showed that PEG was biocompatible with the human placenta with no alteration of the basal rate of proliferation or apoptosis in term placental explants. No significant changes in the released levels of lactate dehydrogenase and the human chorionic gonadotropin were observed after PEG treatment. The cellular uptake studies revealed that conjugating Cy5.5 and haloperidol to PEG significantly reduced (by up to ∼40-fold) their uptake by the placenta. These findings highlight the viability of novel non-prodrug polymer-drug conjugates to avoid the accumulation of drugs within the placenta.

Targeted Delivery of Epidermal Growth Factor to the Human Placenta to Treat Fetal Growth Restriction.

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50-100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery; however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF.

The lexicon of antimicrobial peptides: a complete set of arginine and tryptophan sequences.

Our understanding of the activity of cationic antimicrobial peptides (AMPs) has focused on well-characterized natural sequences, or limited sets of synthetic peptides designed de novo. We have undertaken a comprehensive investigation of the underlying primary structural features that give rise to the development of activity in AMPs. We consider a complete set of all possible peptides, up to 7 residues long, composed of positively charged arginine (R) and / or hydrophobic tryptophan (W), two features most commonly associated with activity. We found the shortest active peptides were 4 or 5 residues in length, and the overall landscapes of activity against gram-positive and gram-negative bacteria and a yeast were positively correlated. For all three organisms we found a single activity peak corresponding to sequences with around 40% R; the presence of adjacent W duplets and triplets also conferred greater activity. The mechanistic basis of these activities comprises a combination of lipid binding, particularly to negatively charged membranes, and additionally peptide aggregation, a mode of action previously uninvestigated for such peptides. The maximum specific antimicrobial activity appeared to occur in peptides of around 10 residues, suggesting 'diminishing returns' for developing larger peptides, when activity is considered per residue of peptide.

Microfluidic-assisted fabrication of phosphatidylcholine-based liposomes for controlled drug delivery of chemotherapeutics.

Microfluidic enables precise control over the continuous mixing of fluid phases at the micrometre scale, aiming to optimize the processing parameters and to facilitate scale-up feasibility. The optimization of parameters to obtain monodispersed drug-loaded liposomes however is challenging. In this work, two phosphatidylcholines (PC) differing in acyl chain length were selected, and used to control the release of the chemotherapeutic agent doxorubicin hydrochloride, an effective drug used to treat breast cancer. Microfluidics was used to rapidly screen manufacturing parameters and PC formulations to obtain monodispersed unilamellar liposomal formulations with a reproducible size (i.e. < 200 nm). Cholesterol was included in all liposomal formulations; some formulations also contained DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine) and/or DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine). Systematic variations in microfluidics total flow rate (TFR) settings were performed, while keeping a constant flow rate ratio (FRR). A total of six PC-based liposomes were fabricated using the optimal manufacturing parameters (TFR 500 µL/min, FRR 0.1) for the production of reproducible, stable liposome formulations with a narrow size distribution. Liposomes actively encapsulating doxorubicin exhibited high encapsulation efficiencies (>80%) for most of the six formulations, and sustained drug release profiles in vitro over 48 h. Drug release profiles varied as a function of the DMPC/DSPC mol content in the lipid bilayer, with DMPC-based liposomes exhibiting a sustained release of doxorubicin when compared to DSPC liposomes. The PC-based liposomes, with a slower release of doxorubicin, were tested in vitro, as to investigate their cytotoxic activity against three human breast cancer cell lines: the non-metastatic ER+/PR + MCF7 cells, the triple-negative aggressive MDA-MB 231 cells, and the metastatic HER2-overexpressing/PR + BT474 cells. Similar cytotoxicity levels to that of free doxorubicin were reported for DMPC5 and DMPC3 binary liposomes (IC50 ~ 1 µM), whereas liposomes composed of a single PC were less cytotoxic (IC50 ~ 3-4 µM). These results highlight that microfluidics is suitable for the manufacture of monodispersed and size-specific PC-based liposomes in a controlled single-step; furthermore, selected PC-based liposome represent promising nanomedicines for the prolonged release of chemotherapeutics, with the aim of improving outcomes for patients.

Functional Evaluation of STOX1 (STORKHEAD-BOX PROTEIN 1) in Placentation, Preeclampsia, and Preterm Birth.

Revaluation of the association of the STOX1 (STORKHEAD_BOX1 PROTEIN 1) transcription factor mutation (Y153H, C allele) with the early utero-vascular origins of placental pathology is warranted. To investigate if placental STOX1 Y153H genotype affects utero-vascular remodeling-compromised in both preterm birth and preeclampsia-we utilized extravillous trophoblast (EVT) explant and placental decidual coculture models, transfection of STOX1 wild-type and mutant plasmids into EVT-like trophoblast cell lines, and a cohort of 75 placentas from obstetric pathologies. Primary EVT and HTR8/SVneo cells carrying STOX1 Y153H secreted lower levels of IL (interleukin) 6, and IL-8, and higher CXCL16 (chemokine [C-X-C motif] ligand 16) and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) than wild-type EVT and Swan71 cells. Media from wild-type EVT or Swan71 cells transfected with wild-type STOX1 stimulated: endothelial chemokine expression, angiogenesis, and decidual natural killer cell and monocyte migration. In contrast, Y153H EVT conditioned medium, Swan71 transfected with the Y153H plasmid, or HTR8/SVneo media had no effect. Genotyping of placental decidual cocultures demonstrated association of the placental STOX1 CC allele with failed vascular remodeling. Decidual GG NODAL R165H increased in failed cocultures carrying the placental CC alleles of STOX1. Multivariate analysis of the placental cohort showed that the STOX1 C allele correlated with premature birth, with or without severe early-onset preeclampsia, and small for gestational age babies. In conclusion, placental STOX1 Y153H is a precipitating factor in preterm birth and placental preeclampsia due to defects in early utero-placental development.

The potential role of the E SRRG pathway in placental dysfunction.

Normal placental development and function is of key importance to fetal growth. Conversely aberrations of placental structure and function are evident in pregnancy complications including fetal growth restriction (FGR) and preeclampsia. Although trophoblast turnover and function is altered in these conditions, their underlying aetiologies and pathophysiology remains unclear, which hampers development of therapeutic interventions. Here we review evidence that supports a role for estrogen related receptor-gamma (ESRRG) in the development of placental dysfunction in FGR and preeclampsia. This relationship deserves particular consideration because ESRRG is highly expressed in normal placenta, is reduced in FGR and preeclampsia and its expression is altered by hypoxia, which is thought to result from deficient placentation seen in FGR and preeclampsia. Several studies have also found microRNA (miRNA) or other potential upstream regulators of ESRRG negatively influence trophoblast function which could contribute to placental dysfunction seen in FGR and preeclampsia. Interestingly, miRNAs regulate ESRRG expression in human trophoblast. Thus, if ESRRG is pivotally associated with the abnormal trophoblast turnover and function it may be targeted by microRNAs or other possible upstream regulators in the placenta. This review explores altered expression of ESRRG and upstream regulation of ESRRG-mediated pathways resulting in the trophoblast turnover, placental vascularisation, and placental metabolism underlying placental dysfunctions. This demonstrates that the ESRRG pathway merits further investigation as a potential therapeutic target in FGR and preeclampsia.

Nanoparticles in pregnancy: the next frontier in reproductive therapeutics.

BACKGROUND: Nanotechnology involves the engineering of structures on a molecular level. Nanomedicine and nano-delivery systems have been designed to deliver therapeutic agents to a target site or organ in a controlled manner, maximizing efficacy while minimizing off-target effects of the therapeutic agent administered. In both reproductive medicine and obstetrics, developing innovative therapeutics is often tempered by fears of damage to the gamete, embryo or developing foetus or of negatively impacting a woman's reproductive potential. Thus, nanomedicine delivery systems may provide alternative targeted intervention strategies, treating the source of the disease and minimizing long-term consequences for the mother and/or her foetus. OBJECTIVE AND RATIONALE: This review summarizes the current state of nanomedicine technology in reproductive medicine and obstetrics, including safety, potential applications, future directions and the hurdles for translation. SEARCH METHODS: A comprehensive electronic literature search of PubMed and Web of Science databases was performed to identify studies published in English up until February 2020. Relevant keywords were used to obtain information regarding use of nanoparticle technology in fertility and gene therapy, early pregnancy complications (ectopic pregnancy and gestational trophoblastic disease) and obstetric complications (preeclampsia, foetal growth restriction, preterm birth and gestational diabetes) and for selective treatment of the mother or foetus. Safety of specific nanoparticles to the gamete, embryo and foetus was also investigated. OUTCOMES: Pre-clinical research in the development of nanoparticle therapeutic delivery is being undertaken in many fields of reproductive medicine. Non-hormonal-targeted nanoparticle therapy for fibroids and endometriosis may provide fertility-sparing medical management. Delivery of interventions via nanotechnology provides opportunities for gene manipulation and delivery in mammalian gametes. Targeting cytotoxic treatments to early pregnancy tissue provides an alternative approach to manage ectopic pregnancies and gestational trophoblastic disease. In pregnancy, nanotherapeutic delivery offers options to stably deliver silencing RNA and microRNA inhibitors to the placenta to regulate gene expression, opening doors to novel genetic treatments for preeclampsia and foetal growth restriction. Restricting delivery of teratogenic drugs to the maternal compartment (such as warfarin) may reduce risks to the foetus. Alternatively, targeted delivery of drugs to the foetus (such as those to treat foetal arrythmias) may minimize side effects for the mother. WIDER IMPLICATIONS: We expect that further development of targeted therapies using nanoparticles in a reproductive setting has promise to eventually allow safe and directed treatments for conditions impacting the health and reproductive capacity of women and for the management of pregnancy and serious pregnancy complications.

Effects of Liposome and Cardiolipin on Folding and Function of Mitochondrial Erv1.

Erv1 (EC number 1.8.3.2) is an essential mitochondrial enzyme catalyzing protein import and oxidative folding in the mitochondrial intermembrane space. Erv1 has both oxidase and cytochrome c reductase activities. While both Erv1 and cytochrome c were reported to be membrane associated in mitochondria, it is unknown how the mitochondrial membrane environment may affect the function of Erv1. Here, in this study, we used liposomes to mimic the mitochondrial membrane and investigated the effect of liposomes and cardiolipin on the folding and function of yeast Erv1. Enzyme kinetics of both the oxidase and cytochrome c reductase activity of Erv1 were studied using oxygen consumption analysis and spectroscopic methods. Our results showed that the presence of liposomes has mild impacts on Erv1 oxidase activity, but significantly inhibited the catalytic efficiency of Erv1 cytochrome c reductase activity in a cardiolipin-dependent manner. Taken together, the results of this study provide important insights into the function of Erv1 in the mitochondria, suggesting that molecular oxygen is a better substrate than cytochrome c for Erv1 in the yeast mitochondria.

Manufacturing drug co-loaded liposomal formulations targeting breast cancer: Influence of preparative method on liposomes characteristics and in vitro toxicity.

Developing more efficient manufacturing methods for nano therapeutic systems is becoming important, not only to better control their physico-chemical characteristics and therapeutic efficacy but also to ensure scale-up is cost-effective. The principle of cross-flow chemistry allows precise control over manufacturing parameters for the fabrication of uniform liposomal formulations, as well as providing reproducible manufacturing scale-up compared to conventional methods. We have herein investigated the use of microfluidics to produce PEGylated DSPC liposomes loaded with doxorubicin and compared their performance against identical formulations prepared by the thin-film method. The isoprenylated coumarin umbelliprenin was selected as a co-therapeutic. Umbelliprenin-loaded and doxorubicin:umbelliprenin co-loaded liposomes were fabricated using the optimised microfluidic set-up. The role of umbelliprenin as lipid bilayer fluidity modulation was characterized, and we investigated its role on liposomes size, size distribution, shape and stability compared to doxorubicin-loaded liposomes. Finally, the toxicity of all liposomal formulations was tested on a panel of human breast cancer cells (MCF-7, MDA-MB 231, BT-474) to identify the most potent formulation by liposomal fabrication method and loaded compound(s). We herein show that the microfluidic system is an alternative method to produce doxorubicin:umbelliprenin co-loaded liposomes, allowing fine control over liposome size (100-250 nm), shape, uniformity and doxorubicin drug loading (>80%). Umbelliprenin was shown to confer fluidity to model lipid biomembranes, which helps to explain the more homogeneous size and shape of co-loaded liposomes compared to liposomes without umbelliprenin. The toxicity of doxorubicin:umbelliprenin co-loaded liposomes was lower than that of free doxorubicin, due to the delayed release of doxorubicin from liposomes. An alternative, rapid and easy manufacturing method for the production of liposomes has been established using microfluidics to effectively produce uniform doxorubicin:umbelliprenin co-loaded liposomal formulations with proven cytotoxicity in human breast cancer cell lines in vitro.

Tetramethoxystilbene-Loaded Liposomes Restore Reactive-Oxygen-Species-Mediated Attenuation of Dilator Responses in Rat Aortic Vessels Ex vivo.

The methylated analogue of the polyphenol resveratrol (RV), 2,3',4,5'-tetramethoxystilbene (TMS) displays potent antioxidant properties and is an effective cytochrome P450 (CYP) 1B1 inhibitor. The bioavailability of TMS is low. Therefore, the use of liposomes for the encapsulation of TMS is a promising delivery modality for enhanced uptake into tissues. We examined the effect of delivery of TMS in liposomes on the restoration of vasodilator responses of isolated aortic vessels after acute tension elevation ex vivo. Aortic vessels from young male Wistar rats were isolated, and endothelial-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) responses assessed. Acute tension elevation (1 h) significantly reduced ACh dilator responses, which were restored following incubation with superoxide dismutase or apocynin (an NADPH oxidase inhibitor). Incubation with TMS-loaded liposomes (mean diameter 157 ± 6 nm; PDI 0.097) significantly improved the attenuated dilator responses following tension elevation, which was sustained over a longer period (4 h) when compared to TMS solution. Endothelial denudation or co-incubation with L-NNA (Nω-nitro-l-arginine; nitric oxide synthase inhibitor) resulted in loss of dilator function. Our findings suggest that TMS-loaded liposomes can restore attenuated endothelial-dependent dilator responses induced by an oxidative environment by reducing NADPH-oxidase-derived ROS and potentiating the release of the vasodilator nitric oxide. TMS-loaded liposomes may be a promising therapeutic strategy to restore vasodilator function in vascular disease.

The role of insulin-like growth factor 2 receptor-mediated homeobox gene expression in human placental apoptosis, and its implications in idiopathic fetal growth restriction.

Fetal growth restriction (FGR) is caused by poor placental development and function early in gestation. It is well known that placentas from women with FGR exhibit reduced cell growth, elevated levels of apoptosis and perturbed expression of the growth factors, cytokines and the homeobox gene family of transcription factors. Previous studies have reported that insulin-like growth factor-2 (IGF2) interacts with its receptor-2 (IGF2R) to regulate villous trophoblast survival and apoptosis. In this study, we hypothesized that human placental IGF2R-mediated homeobox gene expression is altered in FGR and contributes to abnormal trophoblast function. This study was designed to determine the association between IGF2R, homeobox gene expression and cell survival in pregnancies affected by FGR. Third trimester placentas were collected from FGR-affected pregnancies (n = 29) and gestation matched with control pregnancies (n = 30). Functional analyses were then performed in vitro using term placental explants (n = 4) and BeWo trophoblast cells. mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence IGF2R expression in placental explants and the BeWo cell-line. cDNA arrays were used to screen for downstream targets of IGF2R, specifically homeobox gene transcription factors and apoptosis-related genes. Functional effects of silencing IGF2R were then verified by ß-hCG ELISA, caspase activity assays and a real-time electrical cell-impedance assay for differentiation, apoptosis and cell growth potential, respectively. IGF2R expression was significantly decreased in placentas from pregnancies complicated by idiopathic FGR (P < 0.05 versus control). siRNA-mediated IGF2R knockdown in term placental explants and the trophoblast cell line BeWo resulted in altered expression of homeobox gene transcription factors, including increased expression of distal-less homeobox gene 5 (DLX5), and decreased expression of H2.0-Like Homeobox 1 (HLX) (P < 0.05 versus control). Knockdown of IGF2R transcription increased the expression and activity of caspase-6 and caspase-8 in placental explants, decreased BeWo proliferation and increased BeWo differentiation (all P < 0.05 compared to respective controls). This is the first study linking IGF2R placental expression with changes in the expression of homeobox genes that control cellular signalling pathways responsible for increased trophoblast cell apoptosis, which is a characteristic feature of FGR.