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Back to Journals » International Journal of Nanomedicine » Volume 14
In vitro comparison of liposomal drug delivery systems targeting the oxytocin receptor: a potential novel treatment for obstetric complications
Authors Hua S, Vaughan B
Received 13 December 2018
Accepted for publication 9 February 2019
Published 27 March 2019 Volume 2019:14 Pages 2191—2206
DOI https://doi.org/10.2147/IJN.S198116
Editor who approved publication: Prof. Dr. Thomas J. Webster
Susan Hua,1,2 Benjamin Vaughan3
1Therapeutic Targeting Research Group, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia; 2Hunter Medical Research Institute, New Lambton Heights, NSW, Australia; 3Centre for Organic Electronics, University of Newcastle, Callaghan, NSW, Australia
Introduction: Targeted intervention to the uterus has great potential for the treatment of obstetric complications (eg, preterm birth, dysfunctional labor, and postpartum hemorrhage) by improving the effectiveness and safety of therapeutic compounds. In particular, targeting the oxytocin receptor (OTR) is a novel approach for drug delivery to the uterus. The aim of this study was to report the complete data set for the pharmaceutical synthesis and in vitro characterization of PEGylated liposomes conjugated with anti-OTR monoclonal antibodies (OTR-Lipo) or atosiban (ATO-Lipo, OTR antagonist).
Methods: OTR-targeted liposomal platforms composed of 1,2-distearoyl-sn-glycero-2-phosphocholine and cholesterol were prepared according to the method of dried lipid film hydration. Ligands were conjugated with the surface of liposomes using optimized methods to maximize conjugation efficiency. The liposomes were characterized for particle size, ligand conjugation, drug encapsulation, liposome stability, specificity of binding, cellular internalization, mechanistic pathway of cellular uptake, and cellular toxicity.
Results: Both OTR-Lipo and ATO-Lipo showed significant and specific binding to OTRs in a concentration-dependent manner compared to all control groups. There was no significant difference in binding values between OTR-Lipo and ATO-Lipo across all concentrations evaluated. In addition, OTR-Lipo (81.61%±7.84%) and ATO-Lipo (85.59%±8.28%) demonstrated significantly increased cellular internalization in comparison with rabbit IgG immunoliposomes (9.14%±1.71%) and conventional liposomes (4.09%±0.78%) at 2.02 mM phospholipid concentration. Cellular association following liposome incubation at 4.05 mM resulted in similar findings. Evaluation of the mechanistic pathway of cellular uptake indicated that they undergo internalization through both clathrin- and caveolin-mediated mechanisms. Furthermore, cellular toxicity studies have shown no significant effect of both liposomal platforms on cell viability.
Conclusion: This study further supports OTRs as a novel pharmaceutical target for drug delivery. OTR-targeted liposomal platforms may provide an effective way to deliver existing therapies directly to myometrial tissue and avoid adverse effects by circumventing non-target tissues.
Keywords: liposomes, nanoparticles, targeted drug delivery, oxytocin receptor, uterus, obstetric complications
Targeted drug delivery of nanomedicines has shown to be beneficial for increasing the therapeutic index of compounds by improving drug targeting to specific sites of disease and/or attenuating localization in healthy non-target tissues. Obstetrics is an area of clinical medicine that has recently gained attention for drug targeting. Current treatment for obstetric complications (eg, preterm labor, dysfunctional labor, and postpartum hemorrhage) is challenging, as there are limited effective and safe therapeutic options available.1–5 Preterm labor (birth before 37 weeks of gestation) is the most important cause of perinatal morbidity and mortality,6–8 whereas postpartum hemorrhage represents the most important cause of maternal morbidity and mortality worldwide.9,10 In addition, up to one third of women may require induction of labor, primarily due to concerns for the health of the mother or fetus.11,12
The development of novel strategies to deliver therapeutic agents specifically to the uterus would address the clinical challenge of effectively managing these obstetric complications. Ligand-targeted nanomedicines have shown enormous potential for site-specific delivery of therapeutic compounds to designated cell types, which selectively express or overexpress specific receptors at the site of action. For obstetric complications, the smooth muscle layer of the uterus (myometrium) represents an ideal target for pharmacological interventions by controlling the contractility state of the uterus. In this way, therapeutic agents can be directed to the uterus to inhibit myometrial contractions (tocolysis) for the management of preterm labor or enhance myometrial contractions (uterotonic) for the management of dysfunctional labor and postpartum hemorrhage.13
More specifically, the oxytocin receptor (OTR) is considered one of the most important molecules in the myometrium and thus a promising target for drug delivery. OTR agonists and antagonists have been clinically used to modify the contractility of the uterus; however, the clinical efficacy of the available agents has been disappointing. For example, oxytocin was reported to be effective in ~50% of patients,12 and atosiban (ATO-Lipo, OTR antagonist) showed lack of efficacy over other conventional treatments and no evidence of improving neonatal outcomes.14,15 Despite this, OTR density in the uterus significantly increases with the progression of pregnancy.16,17 Low OTR expression has been reported in early gestation, but this rises significantly over the course of pregnancy to ~12-fold by 37–41 weeks.17 Maximal expression is seen after the onset of labor, which is assumed to mediate an increase in sensitivity of the myometrium to oxytocin at term.18,19 Importantly, OTR numbers in the myometrium have also been shown to be upregulated in preterm labor.16,17,20 Therefore, designing a drug delivery system to target this moiety would provide a logical means to enhance local drug delivery to the uterus.
We have recently developed a targeted drug delivery system for the uterus by conjugating anti-OTR polyclonal antibodies to a liposomal platform.21,22 Liposomes have the advantage of having flexible physicochemical and biophysical properties, which allow easy modifications to address different delivery considerations.23–26 The functionality of the drug delivery system was evaluated on murine and human myometrial tissues, as well as in vivo in a murine model of preterm labor.21 However, the polyclonal nature of the antibody posed a significant limitation to specificity of binding to OTRs.22 In order to improve the clinical translation of this technology, it is important to determine the effectiveness of other potential anti-OTR ligands for the targeted delivery system – in particular, anti-OTR monoclonal antibodies and atosiban (OTR antagonist). Therefore, the aim of this study was to report the complete data set for the pharmaceutical synthesis and characterization of PEGylated immunoliposomes conjugated with anti-OTR monoclonal antibodies (OTR-Lipo) and atosiban-conjugated PEGylated liposomes (ATO-Lipo), and compare their specific cellular interaction with OTR-expressing myometrial cells in vitro.
Liposome-related materials
1,2-Distearoyl-sn-glycero-2-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG(2000) maleimide), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy (polyethylene glycol)-2000] (DSPE-PEG(2000) carboxylic acid) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP), cholesterol, atosiban, and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were from Sigma-Aldrich Co. (St Louis, MO, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), CBQCA protein assay kit and N-hydroxysuccinimide (NHS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit IgG antibody and anti-OTR monoclonal antibody (ab181077) were obtained from Abcam (Cambridge, UK). PD-10 column was from VWR International (Radnor, PA, USA). All other chemicals and solvents were of at least analytical grade.
Cell-related materials
FBS, trypsin-EDTA (1:250), trypan blue solution 0.4%, dansylcadaverine (D4008), chlorpromazine hydrochloride (C8138), genistein (G6649), filipin (F9765), methyl-β-cyclodextrin (C4555), nocodazole (M1404), cytochalasin D (C8273), MTT-based assay kit (CGD1), and all buffer reagents were from Sigma-Aldrich Co. L-Glutamine, DMEM, Gibco® Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin), sodium pyruvate, Alexa Fluor™ 488 conjugated cholera toxin subunit B (C34775), and Alexa Fluor 488 conjugated transferrin (T13342) were obtained from Thermo Fisher Scientific. Fluoresbrite® YG carboxylate microspheres (size 1 μm) was from Polysciences Inc. (Warrington, PA, USA). hTERT-immortalized myometrial (hTERT-myo) cells were obtained from Prof Roger Smith (University of Newcastle, Australia). All other reagents were of analytical grade. The POLARstar Optima™ fluorescent plate reader and its corresponding software were obtained from BMG Labtech (Ortenberg, Germany). The Institutional Biosafety Committee at the University of Newcastle approved all experiments and the use of the cell line.
Preparation of immunoliposomes
Immunoliposomes composed of DSPC and cholesterol (molar ratio 2:1) containing DSPE-PEG(2000) maleimide at 1.5 mol% of DSPC as a coupling lipid were prepared according to the method of dried lipid film hydration in PBS (pH 7.4) as previously described.27 DiI (fluorescent lipophilic dye) was added at 0.2 mol% of DSPC, and drugs were incorporated based on their hydrophilicity. The resulting multilamellar dispersions were reduced in size and lamellarity by ultrasonication at 60% amplitude for 5 minutes at 65°C. The activated liposome suspension was immediately mixed with thiolated antibody at room temperature. Thiolated antibodies were prepared by conjugating anti-OTR monoclonal antibodies (25 μg) or non-specific rabbit IgG (25 μg) with SPDP (6.25 mg/mL; SPDP/mAb molar ratio =10:1). PD-10 column equilibrated with distilled water was used to remove excess SPDP and fractions containing pyridyldithiopropionated-Ab (PDP-Ab) conjugates (assessed by absorbance at 280 nm) were lyophilized and stored at 4°C under nitrogen gas. PDP-Ab was reduced with 5 mM TCEP for 5 minutes to produce thiolated-Ab (Ab-SH), and absorbance was checked at 280 nm (protein concentration) and 343 nm (SPDP modification) to ensure stability of the compound. Thiolated antibody was mixed immediately with liposomes for 1 hour at room temperature with stirring in the dark. TLX ultracentrifugation (Optima™) was used to remove unconjugated Ab and non-encapsulated drug (100,000× g; 45 minutes). Liposomes were resuspended in PBS (pH 7.4) and stored at 4°C under nitrogen gas and in the dark, and were used within 2 weeks.
Preparation of atosiban-conjugated liposomes
Liposomes were composed of DSPC, cholesterol, and DSPE-PEG(2000) carboxylic acid at a molar ratio of 2:1:0.06 according to the method of dried lipid film hydration in MES buffer (pH 4.7). The fluorescent lipophilic dye DiI was added at 0.2 mol% of DSPC. Drugs were incorporated based on their hydrophilicity. The resulting multilamellar dispersions were reduced in size and lamellarity by ultrasonication at 60% amplitude for 5 minutes at 65°C. NHS and EDC (molar ratio 2:1) were then added to the liposome suspension and incubated for 15 minutes. Prior to the addition of atosiban, the pH of the liposome suspension was raised to 7.2–7.5 for optimal reaction with amine-containing molecules. The liposome suspension was incubated overnight at room temperature with stirring in the dark. Excess reagents were removed by TLX ultracentrifugation (Optima) (100,000× g; 45 minutes). Liposomes were resuspended in PBS (pH 7.4) and stored at 4°C under nitrogen gas and in the dark and were used within 2 weeks.
Liposome characterization
Size distribution of the liposomes was determined by dynamic laser light scattering (Zetasizer Nano ZS™; ATA Scientific, Taren Point, Australia). Phospholipid concentration was determined indirectly by measuring DiI concentration within liposomes. CBQCA protein assay was used to quantify the amount of antibody associated with the liposomes, using bovine serum albumin for the preparation of the standard curve. Atosiban concentration was evaluated using HPLC.
Drug concentration was determined by HPLC using an Agilent Technologies 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA), consisting of an autoinjector, column oven, binary pump and UV-Vis detector. Data were integrated using Agilent Chemstation software. Separation was performed using a Thermo Scientific BDS Hypersil C18 column (150×4.6 mm, 5 μm). Drugs were dissolved in distilled water (hydrophilic drugs) or ethanol (hydrophobic drugs), with subsequent dilutions made in mobile phase for the calibration curves.
HPLC mobile phase and settings for nifedipine
Mobile phase consisted of 60% acetonitrile and 40% 10 mM KH2PO4 buffer (pH 2.5), which was pumped through the column at 1 mL/min. The column was maintained at 25°C and the wavelength of detection was 208 nm.
HPLC mobile phase and settings for salbutamol hemisulfate
Mobile phase consisted of 30% methanol and 70% 0.05 M KH2PO4 buffer (pH 3.5), which was pumped through the column at 1 mL/min. The column was maintained at 25°C and the wavelength of detection was 230 nm.
HPLC mobile phase and settings for atosiban
Mobile phase consisted of 30% acetonitrile and 70% 10 mM KH2PO4 (pH 2.5), which was pumped through the column at 1 mL/min. The column was maintained at 25°C and the wavelength of detection was 220 nm.
Atomic force microscopy (AFM)
AFM was performed on a Cypher Scanning Probe Microscope (Asylum Research; Oxford Instruments, Abingdon, UK). Commercial pyramidal silicon tips (TAPAl300-G; BudgetSensors, Sofia, Bulgaria) with a radius <10 nm, a resonance frequency of ~300 kHz and a nominal force constant of 40 (20–75) N/m were used. All measurements were performed in tapping mode to avoid damage of the sample surface. The scan speed was proportional to the scan size and the scan rate was between 1.95 and 2.44 Hz. The results were visualized in amplitude mode. Liposomes were directly transferred onto a silicon chip by dipping it into the liposome suspension. The silicon chip was then dried at room temperature before analysis.
Liposome stability assay
The dialysis technique was used to evaluate the in vitro stability of the liposomal formulations in PBS pH 7.4% and 50% FBS at 37°C over a 48 hours duration, as previously described.22 A modified assay was used to evaluate the true release of each drug from the liposomes without surpassing saturation point, which addressed the potential solubility issues across the dialysis membrane. Drug retention percentage was determined by: Drug retention (%) = 100% − [(Dt/D0) × 100%], where Dt and D0 indicate the amount of drug released from the liposomes at certain intervals and the total amount of drug in the liposome suspension, respectively. Liposome samples were collected at the end of the study and lysed with ethanol for analysis of drug content by HPLC.
Specificity of binding to OTRs on myometrial cells
Specificity of binding was evaluated on hTERT-myo cells as previously described.22 In brief, cells were seeded at 4×104 cells per well in 96-well tissue culture plates in complete medium (DMEM containing 1% L-glutamine, 1% sodium pyruvate, 1% Gibco Antibiotic-Antimycotic, 10% FBS) at 37°C in 5% CO2. At confluence, the plates were exchanged with serum and supplement-free DMEM to avoid binding of serum proteins to the liposome surfaces which might induce agglomeration. For competitive inhibition studies, cells were preincubated with a saturating concentration of anti-OTR monoclonal antibodies for 30 minutes. Liposomes were added to the cells for 1 hour at 4°C to determine exclusively cell binding.28,29 A fluorescent dye (DiI) was incorporated into the phospholipid bilayer as a marker for the liposomes. At the end of incubation, ice-cold PBS was used to wash the cells three times to remove unbound liposomes and fluorimetric detection (POLARstar Optima) was used to assess binding at excitation/emission wavelengths corresponding to DiI (549/565 nm). Background reading was assessed with cells incubated in medium alone.
Cellular uptake of OTR-targeted liposomes
Cellular association of OTR-targeted liposomes was assessed on hTERT-myo cells as previously described.22 In brief, cells were seeded in 96-well tissue culture plates (4×104 cells per well) and grown until confluent under the conditions described above. At confluence, the plates were exchanged with serum and supplement-free DMEM prior to the addition of fluorescent-labeled liposomes for 1 hour at 37°C. It should be noted that both receptor binding and internalization takes place at this temperature.28,29 At the end of incubation, ice-cold PBS was used to wash the cells three times before fluorimetric detection to assess extracellularly bound liposomes. Acid wash (0.1 M HCl for 10 minutes)30 was used to release surface-bound fluorescence before lysis of the cells with the addition of 1% Triton X-100 in PBS for 10 minutes at 37°C. Results from the acid wash were expressed as extracellularly bound fluorescence, whereas fluorescence in the lysis fractions was expressed as internalized fluorescence. Background reading was evaluated with solubilized cells without prior incubation with liposomes.
Endocytotic mechanisms of OTR-targeted liposomes
Mechanisms of cellular uptake were determined in the presence of specific inhibitory agents for different types of endocytosis, as previously described.22 In brief, hTERT-myo cells were preincubated with specific inhibitory agents for 30 minutes at 37°C and were then incubated with liposomes for 1 hour at 37°C. Chlorpromazine hydrochloride and monodansylcadaverine were used as inhibitiors of clathrin-mediated endocytosis, whereas genistein and filipin were used to inhibit caveolin-mediated endocytosis. Methyl-β-cyclodextrin is able to inhibit both clathrin- and caveolin-mediated endocytosis by depleting cholesterol. Nocodazole was used to inhibit macropinocytosis, and cytochalasin D was used to inhibit both phagocytosis and macropinocytosis. Positive controls were used together with specific inhibitors to investigate clathrin-mediated endocytosis (Alexa Fluor 488 conjugated transferrin at 120 μg/mL), caveolin-mediated endocytosis (Alexa Fluor 488 conjugated cholera toxin subunit B at 0.6 μg/mL), and phagocytosis and macropinocytosis (Fluoresbrite YG carboxylate microspheres of 1 μm diameter at 100 μg/mL). At the end of incubation, cells were washed three times with ice-cold PBS before fluorimetric detection as described above.
Cellular toxicity
Cell viability following exposure of hTERT-myo cells to liposomes and specific endocytotic inhibitors at various concentrations for 24 and 1.5 hour, respectively, was measured using an MTT-based assay, as previously described.22 Triton X-100 (1% in PBS) was used as a positive control. At the end of incubation, the medium was removed and MTT solution (5 mg/mL MTT in supplemented DMEM) was added to the cultures (final concentration of 0.5 mg/mL) and incubated for 4 hours at 37°C. The medium was then discarded and formazan crystals were solubilized using 150 μL dimethylsulfoxide for 15 minutes under light protection and at room temperature. Absorbance was measured at 550 nm in a microplate spectrophotometer (POLARstar Optima), which is directly proportional to cellular metabolism. Background reading was assessed with untreated cells.
All data are expressed as mean ± standard error of the mean (SEM) or SD. GraphPad Prism 7.01 software was used for statistical analysis. One-way ANOVA with Tukey's multiple comparison test was used to evaluate differences between formulation groups or between time points (one independent variable). Comparisons between the different formulation groups over various concentrations were made using two-way ANOVA with Tukey's multiple comparison test (two independent variables). Differences were considered significant when P<0.05.
Dispersion properties of the liposome formulations
OTR-Lipo and ATO-Lipo had a mean particle size of 201±2.9 and 201±3.8 nm with a polydispersity index of 0.240±0.072 and 0.220±0.056, respectively. The size and polydispersity index of the control and drug-encapsulated liposome formulations were similar (Table 1). The choice of particle size was based on the results from our previous studies.21,22 The low polydispersity indexes indicate that the mean particle size is a reasonable indicator of the size of the majority of the particles in the dispersions. All liposome formulations had an approximate neutral net charge, as they were composed of neutral phospholipids. Both nifedipine-loaded liposomes and salbutamol-loaded liposomes had drug encapsulation efficiencies of >98%, which equated to ~4 mg of drug per milliliter of liposome suspension (Table 1). Mean atosiban coupling ratio for the ATO-Lipo was 5.970±0.232 μg of peptide per μmol of phospholipid, which equated to ~2,017±78.39 molecules per liposome. Mean antibody coupling ratio for the OTR-Lipo was 1.227±0.023 μg of antibody per μmol of phospholipid. With a starting antibody concentration of 25 μg and a phospholipid concentration of 2.026×10−5 mol, this equated to a conjugation efficiency of >99% (Table 1). Experiments were conducted to monitor size and size distribution of the liposome formulations under storage conditions of 4°C in PBS pH 7.4 over 12 weeks to ensure stability over time. The size and polydispersity index of the liposome formulations were stable over this period (Figure 1).
Table 1 Physicochemical characteristics of liposome formulations (mean ± SD, n=3)
Abbreviations: OTR-Lipo, PEGylated immunoliposomes conjugated with anti-oxytocin receptor monoclonal antibodies; ATO-Lipo, atosiban-conjugated PEGylated liposomes.
Figure 1 Particle size and polydispersity index of OTR-Lipo (A, B) and ATO-Lipo (C, D) over a period of 12 weeks.
Note: The results are represented as mean ± SD of three independent experiments (P>0.05, ANOVA).
Stability of drug-encapsulated OTR-targeted liposomes
Nifedipine (hydrophobic drug) and salbutamol hemisulfate (hydrophilic drug) were used to evaluate the in vitro stability of OTR-Lipo and ATO-Lipo. Nifedipine is a hydrophobic compound; therefore, its solubility in PBS pH 7.4 was initially assessed to ensure that the parameters of the dialysis assay did not surpass the saturation point for the drug. This determined that 70 μg of nifedipine in 10 mL PBS solution within the dialysis tubing was able to dialyze into 30 mL of the PBS release volume. This was not required for salbutamol as it is readily soluble in an aqueous phase, thereby allowing 1 mg of the drug to be placed in 10 mL PBS solution within the dialysis tubing. Minimal drug release was detected for both salbutamol-loaded OTR-targeted liposomes and nifedipine-loaded OTR-targeted liposomes in PBS at 37°C across the 48 hours duration of the experiment (Figure 2). In serum, there was a significant difference in stability at 24 and 48 hours compared to baseline values for both the liposome formulations (Figure 2). This equated to a decrease of 19.7% for nifedipine-loaded OTR-Lipo (P<0.01), 23.9% for salbutamol-loaded OTR-Lipo (P<0.01), 26.2% for nifedipine-loaded ATO-Lipo (P<0.0001), and 19.2% for salbutamol-loaded ATO-Lipo (P<0.01) at 48 hours. Drug release was not limited by the diffusion of free drug through the dialysis membrane, as shown by complete recovery of the drug solutions in the dialysis medium within 6 hours. Therefore, both of the drugs evaluated were able to pass through the cellulose membrane freely.
Figure 2 Stability of nifedipine-loaded OTR-Lipo (A), nifedipine-loaded ATO-Lipo (B), salbutamol-loaded OTR-Lipo (C), and salbutamol-loaded ATO-Lipo (D) in PBS pH 7.4 and 50% FBS at 37°C.
Notes: The data represent the mean ± SD of three independent experiments. One-way ANOVA with Tukey's multiple comparison test was used to assess drug retention at various time points compared to their respective baseline values (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Liposomal imaging with AFM
AFM was used to visualize all liposomal formulations when dried to confirm particle size and morphology. For size determination, all visible particles within a representative scan area were individually evaluated. OTR-Lipo, ATO-Lipo, rabbit IgG immunoliposomes, and conventional liposomes showed approximate vesicle sizes of 200 nm (Figure 3), which corresponds with the results from dynamic laser light scattering. The surface of conventional liposomes was typically smooth (Figure 3D), whereas small surface structures can be detected at the membrane border for OTR-Lipo (Figure 3A) and rabbit IgG immunoliposomes (Figure 3C) and only slightly for ATO-Lipo (Figure 3B). Surface coverage in the height mode was difficult to evaluate due to the collapsed vesicular structure when dried.
Figure 3 Representative AFM images of OTR-Lipo (A), ATO-Lipo (B), rabbit IgG immunoliposomes (C), and conventional liposomes (D).
Note: Scale bars correspond to 50 nm.
Abbreviations: AFM, atomic force microscopy; OTR-Lipo, PEGylated immunoliposomes conjugated with anti-oxytocin receptor monoclonal antibodies; ATO-Lipo, atosiban-conjugated PEGylated liposomes.
Specificity of binding
OTR-Lipo and ATO-Lipo bound significantly to hTERT-myo cells in a concentration-dependent manner in comparison with all control groups (P<0.0001) (Figure 4). There was no significant difference in binding values between OTR-Lipo and ATO-Lipo across all concentrations evaluated (P>0.05). Negative control binding experiments using non-specific rabbit-IgG immunoliposomes and conventional liposomes were assessed to demonstrate the specificity of the binding toward OTR expressing cells. The results showed very low cell binding values across all concentrations evaluated and were not significantly different (P>0.05). In another set of experiments, hTERT-myo cells were pretreated with excess monoclonal antibody directed against OTRs to further demonstrate the specific nature of the OTR-Lipo and ATO-Lipo interaction. Figure 4 shows significant inhibition of binding of both OTR-Lipo and ATO-Lipo to hTERT-myo cells pretreated with excess anti-OTR monoclonal antibody (1 mg/mL) by up to 95.0% (P<0.0001) and 92.1% (P<0.0001), respectively.
Figure 4 Concentration-dependent binding of OTR-Lipo and ATO-Lipo to hTERT-myo cells.
Notes: Conventional liposomes and rabbit IgG immunoliposomes were used as controls. Competitive inhibition of binding of OTR-targeted liposomes was also evaluated following preincubation with excess anti-OTR monoclonal antibody. Liposomes were incubated for 1 hour at 4°C. The results are represented as mean ± SEM of six independent experiments. Two-way ANOVA with Tukey's multiple comparison test was used to assess intergroup differences at various concentrations (****P<0.0001).
Abbreviations: SEM, standard error of the mean; OTR-Lipo, PEGylated immunoliposomes conjugated with anti-oxytocin receptor monoclonal antibodies; ATO-Lipo, atosiban-conjugated PEGylated liposomes.
Degree of cellular uptake
OTR-Lipo (81.61%±7.84%) and ATO-Lipo (85.59%±8.28%) demonstrated significantly increased cellular internalization compared with rabbit IgG immunoliposomes (9.14%±1.71%) and conventional liposomes (4.09%±0.78%) at 2.02 mM phospholipid concentration after 1 hour of incubation at 37°C (Figure 5A, P<0.0001). Similar results were shown following liposome incubation at 4.05 mM (Figure 5B), with cell-bound liposomes of 15.95%±2.45% and 9.21%±0.74% and cellular internalization of 84.05%±12.30% and 85.60%±11.00% for OTR-Lipo and ATO-Lipo, respectively. Low levels of cell binding were shown for non-specific rabbit-IgG immunoliposomes and conventional liposomes at both concentrations, which were not significantly different from cell-bound OTR-Lipo and ATO-Lipo (P>0.05). No significant difference was also detected between cellular binding and uptake for rabbit-IgG immunoliposomes and conventional liposomes (P>0.05).
Figure 5 Cellular association of OTR-Lipo, ATO-Lipo, rabbit IgG immunoliposomes and conventional liposomes by hTERT-myo cells.
Notes: Liposomes were incubated at 2.02 mM (A) and 4.05 mM (B) for 1 hour at 37°C. The results are represented as mean ± SEM of six independent experiments. Two-way ANOVA with Tukey's multiple comparison test was used to assess intergroup differences in cell binding and internalization, as well as individual group differences between cell binding and internalization (****P<0.0001).
Mechanisms of cellular uptake
All three pharmacological inhibitors of clathrin-mediated endocytosis (chlorpromazine, monodansylcadaverine, and methyl-β-cyclodextrin) were able to significantly block the internalization of OTR-Lipo and ATO-Lipo (Figure 6, P<0.0001). As further support of the specificity for inhibition of clathrin-mediated endocytosis, the results also showed significant inhibition of uptake for fluorescent-labeled transferrin with all three compounds (positive control, P<0.0001). However, methyl-β-cyclodextrin is also known to block caveolin-mediated endocytosis, which is supported by significant inhibition of uptake of the positive control – cholera toxin subunit B (Figure 6C, P<0.0001). Hence, further investigations were conducted with more specific pharmacological inhibitors of caveolin-mediated endocytosis (genistein and filipin). Figure 7A shows significant inhibition of cellular uptake for OTR-Lipo (P<0.01), ATO-Lipo (P<0.0001), and fluorescent-labeled cholera toxin subunit B (positive control, P<0.001) with genistein. Filipin did not affect the uptake of both OTR-targeted liposomes or cholera toxin subunit B on hTERT-myo cells (Figure 7B, P>0.05). Furthermore, no significant inhibition of cellular uptake was observed for OTR-Lipo and ATO-Lipo with cytochalasin D (Figure 7C, P>0.05), a pharmacological inhibitor of both phagocytosis and macropinocytosis. Cytochalasin D was, however, able to inhibit the uptake of fluorescent microspheres of 1 μm diameter (positive control, P<0.0001). Nocodazole (pharmacological inhibitor of macropinocytosis) was unable to block the cellular uptake of both OTR-targeted liposomes and positive control (fluorescent microspheres) across all concentrations evaluated (Figure 7D, P>0.05).
Figure 6 Cellular uptake of OTR-Lipo and ATO-Lipo following preincubation with various concentrations of pharmacological inhibitors of clathrin-mediated endocytosis (A, B and C).
Notes: Liposomes were incubated at 2.02 mM for 1 hour at 37°C. The results are represented as mean ± SEM of six independent experiments. One-way ANOVA with Tukey's multiple comparison test was used to assess inhibition of cellular uptake of OTR-targeted liposomes and positive control (transferrin) in the presence of various concentrations of inhibitors compared to their respective baseline values (**P<0.01, ***P<0.001, ****P<0.0001).
Figure 7 Cellular uptake of OTR-Lipo and ATO-Lipo following preincubation with various concentrations of pharmacological inhibitors of caveolin-mediated endocytosis (A and B) and macropinocytosis and phagocytosis (C and D).
Notes: Liposomes were incubated at 2.02 mM for 1 hour at 37°C. The results are represented as mean ± SEM of six independent experiments. One-way ANOVA with Tukey's multiple comparison test was used to assess inhibition of cellular uptake of OTR-targeted liposomes and positive control (cholera toxin subunit B or 1 μm microspheres) in the presence of various concentrations of inhibitors compared to their respective baseline values (**P<0.01, ***P<0.001, ****P<0.0001).
Cell viability studies were conducted following incubation with liposomes and the endocytotic inhibitors to rule out cellular toxicity as a potential reason for decreased liposome internalization during these experiments (Figure 8). The results showed no significant effect of OTR-Lipo and ATO-Lipo on the viability of hTERT-myo cells following 24 hours of incubation at both the concentrations evaluated (2.02 and 4.05 mM) (P>0.05). Furthermore, incubation with the endocytic inhibitors at the concentrations used in the in vitro experiments for 1.5 hour (cellular incubation time) did not impair cell viability (P>0.05). Significant cell death was demonstrated for hTERT-myo cells treated with Triton X-100 (positive control) (P<0.0001).
Figure 8 Cell viability following exposure to OTR-targeted liposomes and specific endocytotic inhibitors at various concentrations for 24 and 1.5 hour, respectively.
Notes: Triton X-100 (1% in PBS) was used as a positive control. Two-way ANOVA with Tukey's multiple comparison test was used to assess cell viability at various concentrations compared to healthy control cells (****P<0.0001).
We have engineered liposomal platforms that are able to specifically target OTRs expressed on myometrial cells. OTRs are cell surface receptors containing seven transmembrane domains and belong to the class I family of G protein-coupled receptors.31 They are regulated by changes in receptor expression and desensitization, as well as local changes in oxytocin concentration.11,31–33 The expression of OTRs in the uterus is significantly upregulated during gestation and undergoes rapid downregulation after parturition.11,16,17 Importantly, OTR levels were shown to be maximal and significantly higher after the onset of labor, either preterm or term, than before the onset of labor.17 This tissue-specific regulation of OTR expression is ideal for specific and local accumulation of OTR-targeted nanoparticles to the uterus in obstetric complications such as preterm labor, dysfunctional labor and postpartum hemorrhage21 – in which the management of these conditions is challenging due to limited effective and safe treatment options.1–5 In the uterus, the expression of OTRs is not confined to the myometrium. Chorio-decidual tissue expression of OTRs also increases during parturition34 and spatial differences exist between OTR expression within uterine tissues, which is suggested to aid fetal decent and passage during labor.11
When anti-OTR monoclonal antibodies or rabbit IgG molecules were conjugated to the liposomes, small surface structures were visible along the membrane border. Based on molecular weights of the targeting ligands, we would not expect to see the conjugation of atosiban with the surface of liposomes as clearly as compared with monoclonal antibodies or IgG molecules. Studies in the literature have attempted to visualize the conjugation of targeting ligands to the surface of liposomes using AFM with varying results.35,36 For example, Bendas et al demonstrated that when antibodies are attached to liposomes via a PEG 2000 anchor, it can cause high protein mobility that affects the ability to image the proteins,36 whereas Anabousi et al reported the detection of small globular structures at the surface of such liposomes.35 Similar to Bendas et al,36 we found it difficult to clearly image the anti-OTR antibodies attached to the liposomes via a PEG 2000 anchor. Data obtained from AFM can also deviate slightly from the results of dynamic laser light scattering because of an interaction of the soft and flexible liposomes with the surface of the silicon chip substrate, which we also observed. This is particularly relevant when the liposomes are dried onto the silicon chip prior to analysis, thereby resulting in liposomes flattening and/or spreading on the silicon support.
Conjugation of OTR targeting ligands to the surface of liposomes increased cellular interaction with myometrial uterine cells. hTERT-immortalized myometrial cells were used to evaluate specificity of OTR targeting, as they exhibit the phenotype and properties of highly differentiated smooth muscle cells, without the high variability of responses seen with primary myometrial cells.37,38 Human myometrial cells express OTRs39–41 and have been reported to acquire additional OTRs in culture,42–44 with upregulation likely due to the presence of fetal bovine serum in the culture medium.45 It should be noted that serum and supplement free media were used in the in vitro cellular association studies to determine binding and cellular uptake of the OTR-targeted liposomal platforms in a reductionist in vitro system, which would mimic the in vivo situation of when the liposomes reached the myometrium of the uterus. We have demonstrated highly specific binding of OTR-Lipo and ATO-Lipo to OTR-expressing hTERT-myo cells using concentration-dependent binding studies, competitive inhibition experiments, and negative control binding studies. No significant difference was detected in binding efficiency between liposomes conjugated with anti-OTR monoclonal antibodies and atosiban. Similarly, Refuerzo et al showed improved attachment, retention, and targeting efficiency with atosiban-conjugated liposomes compared with non-targeted liposomes on smooth muscle cells isolated from pregnant humans and mice.46 The use of monoclonal antibodies is preferential to polyclonal antibodies for translational purposes to improve the specificity of binding. This is due to monoclonal antibodies being able to specifically detect a particular epitope on the antigen, which means they are less likely to cross-react with other proteins.
Cellular association experiments showed significant cellular uptake for both OTR-Lipo and ATO-Lipo following binding, which is necessary for subsequent intracellular processing and release of encapsulated therapeutic agents in myometrial cells. Interestingly, anti-OTR monoclonal antibodies and atosiban in the free drug form typically act by blocking natural ligands from binding to myometrial OTRs on the membrane surface.14,15 However, conjugation of these ligands to the surface of liposomes enhances its uptake into myometrial cells. The cellular interaction of liposomes is highly dependent on several liposomal characteristics including particle size, charge, composition, sterical stabilization, and specific properties of the conjugated ligands.23,27 OTR-targeted liposomes themselves are unlikely to impact on OTR desensitization rates, as the concentration of ligand conjugated to the surface of the liposomes is low. Usually much higher doses and continuous dosing of oxytocin are required to cause downregulation of OTRs.47–49
Evaluation of the mechanistic pathway of cellular uptake of the different OTR-targeted liposomal platforms indicates that they both undergo internalization through clathrin- and caveolin-mediated mechanisms. OTRs have previously been shown to congregate with β-arrestin following activation into defined punctuated regions of the plasma membrane, which suggest that they are targeted into clathrin-coated pits for internalization.31,50 OTRs are also highly expressed in the cholesterol-rich, caveolin-containing membrane domains of the plasma membrane.51,52 Furthermore, results from cellular toxicity experiments showed no significant effects of both OTR-targeted liposomal platforms and the endocytotic inhibitors on cell viability at 24 and 1.5 hour (cellular incubation time), respectively. It should be noted that utilization of pharmacological inhibitors is a common approach for studying endocytotic pathways. Generally, a number of pharmacological inhibitors should be evaluated as they can lack specificity for defined pathways and have been shown to be cell type dependent.53 Therefore, a balance is needed between the concentration of inhibitor high enough to inhibit endocytosis but not to induce cytotoxicity.53
OTR-Lipo and ATO-Lipo were shown to be highly stable upon dilution in both an aqueous phase (PBS pH 7.4) and serum (50% FBS). Drug retention was only significantly lower than baseline values when the liposomes were dispersed in serum at 24 and 48 hours. This is likely due to the bioactive substance in FBS, including plasma proteins which can lead to opsonization, destabilization, and lipid exchange.23 The amount of encapsulated drug released was between 19.2% and 26.2% at 48 hours. This is unlikely to affect the performance of the OTR-targeted liposomes, as human serum generally contains 7% proteins (50% FBS was used in the experiment), and the expected application for the technology would require good stability in the circulation particularly following the first few hours after initial administration. Therefore, the targeted nanoparticles are suitable for administration to sites of high dilution, which occurs following intravenous administration. Stability against leakage has been achieved by using phospholipids (eg, DSPC) that remain in the solid phase at physiological temperatures and incorporating cholesterol in the lipid bilayer to minimize lipid exchange.54 PEGylation also improves the circulation half-life of the formulation by evading recognition by the immune system.23,55,56 Salbutamol and nifedipine were chosen for the stability study due to their difference in hydrophobicity, which allows evaluation of the delivery system to potentially encapsulate a variety of therapeutic agents. Hydrophobic molecules are inserted into the bilayer membrane, and hydrophilic molecules can be entrapped in the aqueous center.23 In addition, salbutamol and nifedipine are currently used to treat preterm labor and have been associated with significant maternal and fetal adverse effects, due to the high doses needed to achieve therapeutic effects in the uterus.2–4
The functionality of the OTR-targeted liposomal platforms has previously been evaluated on murine and human myometrial tissues as well as in vivo in a murine model of preterm labor.21,46 We have shown that liposomes conjugated with anti-OTR polyclonal antibodies (200 nm) and loaded with salbutamol, nifedipine, or rolipram (tocolytic agents) inhibited myometrial contractions ex vivo, while those loaded with dofetilide (uterotonic agent) increased contraction duration.21 Empty OTR-targeted liposomes and non-targeted control liposomes loaded with these agents had no effect.21 Similar findings have also been demonstrated for ATO-Lipo (124 nm).46 Furthermore, liposomes loaded with indomethacin (tocolytic agent) and conjugated with anti-OTR polyclonal antibodies21 or atosiban46 significantly reduced the rates of preterm birth in mice compared with non-targeted liposomes and showed no detectable accumulation in the maternal brain or fetus.21,46 Parturition is considered an inflammatory process,23,57 which indicates that the enhanced permeability and retention (EPR) effect may also play a role in liposome accumulation into the uterus. To what extent EPR occurs in parturition as well as in obstetric complications will need to be evaluated in future studies. It should be noted, however, that OTR-targeted liposomes are unlikely to be effective in patients previously exposed to continuous high doses of oxytocin, as this typically leads to desensitization of OTRs for hours or even days,47,48 thereby reducing the targeting effectiveness of the drug delivery system. This phenomenon is accompanied by a downregulation of OTRs at the protein and mRNA level.47,49
This study further supports OTRs as a novel pharmaceutical target for drug delivery. OTR-targeted liposomes may provide an effective way to deliver existing therapies directly to myometrial tissue for the treatment of obstetric complications and avoid adverse effects by circumventing non-target tissues. Achieving targeted delivery of therapeutic agents to the myometrium would potentially increase therapeutic efficacy, decrease the therapeutically effective dose, and/or reduce the risk of systemic side effects. This study shows that when all physicochemical parameters of the liposomes are the same, both OTR-Lipo and ATO-Lipo have similar stability, specificity in OTR binding, degree of cellular uptake, mechanism of endocytotic uptake, and effect on cell viability. Translational development will depend on comprehensive preclinical and clinical studies, as well as other factors specific to the commercialization of nanomedicines.58
In addition to the uterus, OTRs have been reported to be expressed in a multitude of tissues, including breast, pituitary, kidney, ovary, thymus, heart, vascular endothelium, osteoclasts, myoblasts, pancreatic islet cells, and adipocytes.11,31 However, their clinical relevance in humans has not been fully established. Further studies will be required to quantify the biodistribution of OTR-targeted liposomes in these tissues in relevant animal models of these obstetric complications. OTRs are also structurally similar to the vasopressin receptor subtypes.11,20,59 It will be important to determine the degree of cross-reactivity of the different OTR-targeted liposomal platforms with vasopressin receptors and the potential clinical implications of this interaction with regard to biodistribution of the delivery system. It should be noted that vasopressin receptors (V1a receptors) are also expressed in the uterus and are functionally coupled to myometrial contraction.60,61 Interestingly, OTRs have also been detected in several human cancer tissues and cell lines,31 including human breast cancer,62,63 uterine leiomyoma,41,64 adenocarcinoma of the endometrium,65 neuroblastoma, and glioma.66 Therefore, OTR-targeted drug delivery systems may also be adapted to assist in the treatment of other disease pathologies characterized by an upregulation of OTR expression.
This work was supported by the National Health and Medical Research Council, Hunter Medical Research Institute, Global Alliance to Prevent Prematurity and Stillbirth, Gladys M Brawn Fellowship, and University of Newcastle. This work was performed in part at the Materials node of the Australian National Fabrication Facility, which is a company established under the National Collaborative Research Infrastructure Strategy to provide nano and microfabrication facilities for Australian researchers.
SH contributed to study concept and design, liposome manufacturing and characterization, liposome imaging, cellular studies, analysis and interpretation of data, and drafting of manuscript. BV contributed to liposome imaging and analysis and interpretation of data. Both authors were involved in revising the article critically for important intellectual content and gave final approval of the version to be published. The authors agree to be accountable for all aspects of the work.
SH has a patent through the University of Newcastle related to the use of targeted liposomes. BV reports no conflicts of interest in this work.
Butwick AJ, Carvalho B, Blumenfeld YJ, El-Sayed YY, Nelson LM, Bateman BT. Second-line uterotonics and the risk of hemorrhage-related morbidity. Am J Obstet Gynecol. 2015;212(5):642.e1–642.e7.
de Heus R, Mol BW, Erwich JJ, et al. Adverse drug reactions to tocolytic treatment for preterm labour: prospective cohort study. BMJ. 2009;338:b744.
Haas DM, Caldwell DM, Kirkpatrick P, Mcintosh JJ, Welton NJ. Tocolytic therapy for preterm delivery: systematic review and network meta-analysis. BMJ. 2012;345:e6226.
Jørgensen JS, Weile LK, Lamont RF. Preterm labor: current tocolytic options for the treatment of preterm labor. Expert Opin Pharmacother. 2014;15(5):585–588.
Mousa HA, Blum J, Abou El Senoun G, Shakur H, Alfirevic Z. Treatment for primary postpartum haemorrhage. Cochrane Database Syst Rev. 2014;(2):CD003249.
Liu L, Oza S, Hogan D, et al. Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis. Lancet. 2015;385(9966):430–440.
Manuck TA, Rice MM, Bailit JL, et al. Preterm neonatal morbidity and mortality by gestational age: a contemporary cohort. Am J Obstet Gynecol. 2016;215(1):103.e1–103.e14.
Romero R, Dey SK, Fisher SJ. Preterm labor: one syndrome, many causes. Science. 2014;345(6198):760–765.
Al-Zirqi I, Vangen S, Forsen L, Stray-Pedersen B. Prevalence and risk factors of severe obstetric haemorrhage. BJOG. 2008;115(10):1265–1272.
Khan KS, Wojdyla D, Say L, Gülmezoglu AM, van Look PF. Who analysis of causes of maternal death: a systematic review. Lancet. 2006;367(9516):1066–1074.
Arrowsmith S, Wray S. Oxytocin: its mechanism of action and receptor signalling in the myometrium. J Neuroendocrinol. 2014;26(6):356–369.
Blanch G, Lavender T, Walkinshaw S, Alfirevic Z. Dysfunctional labour: a randomised trial. Br J Obstet Gynaecol. 1998;105(1):117–120.
Arrowsmith S, Kendrick A, Wray S. Drugs acting on the pregnant uterus. Obstet Gynaecol Reprod Med. 2010;20(8):241–247.
Goodwin TM, Paul R, Silver H, et al. The effect of the oxytocin antagonist atosiban on preterm uterine activity in the human. Am J Obstet Gynecol. 1994;170(2):474–478.
Romero R, Sibai BM, Sanchez-Ramos L, et al. An oxytocin receptor antagonist (atosiban) in the treatment of preterm labor: a randomized, double-blind, placebo-controlled trial with tocolytic rescue. Am J Obstet Gynecol. 2000;182(5):1173–1183.
Wathes DC, Borwick SC, Timmons PM, Leung ST, Thornton S. Oxytocin receptor expression in human term and preterm gestational tissues prior to and following the onset of labour. J Endocrinol. 1999;161(1):143–151.
Fuchs AR, Fuchs F, Husslein P, Soloff MS. Oxytocin receptors in the human uterus during pregnancy and parturition. Am J Obstet Gynecol. 1984;150(6):734–741.
Fuchs AR, Fields MJ, Freidman S, Shemesh M, Ivell R. Oxytocin and the timing of parturition. Influence of oxytocin receptor gene expression, oxytocin secretion, and oxytocin-induced prostaglandin F2 alpha and E2 release. Adv Exp Med Biol. 1995;395:405–420.
Kimura T, Takemura M, Nomura S, et al. Expression of oxytocin receptor in human pregnant myometrium. Endocrinology. 1996;137(2):780–785.
Akerlund M. Targeting the oxytocin receptor to relax the myometrium. Expert Opin Ther Targets. 2006;10(3):423–427.
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(3):283.e1–283.e14.
Hua S. Synthesis and in vitro characterization of oxytocin receptor targeted PEGylated immunoliposomes for drug delivery to the uterus. J Liposome Res. 2019:1–11.
Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and challenges of liposome assisted drug delivery. Front Pharmacol. 2015;6:286.
Ulrich AS. Biophysical aspects of using liposomes as delivery vehicles. Biosci Rep. 2002;22(2):129–150.
Majumder P, Bhunia S, Bhattacharyya J, Chaudhuri A. Inhibiting tumor growth by targeting liposomally encapsulated CDC20siRNA to tumor vasculature: therapeutic RNA interference. J Control Release. 2014;180:100–108.
Reddy JA, Abburi C, Hofland H, et al. Folate-targeted, cationic liposome-mediated gene transfer into disseminated peritoneal tumors. Gene Ther. 2002;9(22):1542–1550.
Hua S, Chang HI, Davies NM, Cabot PJ. 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. J Liposome Res. 2011;21(2):95–105.
Lopes de Menezes DE, Pilarski LM, Allen TM. In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma. Cancer Res. 1998;58(15):3320–3330.
Voinea M, Manduteanu I, Dragomir E, Capraru M, Simionescu M. Immunoliposomes directed toward VCAM-1 interact specifically with activated endothelial cells-a potential tool for specific drug delivery. Pharm Res. 2005;22(11):1906–1917.
Everts M, Koning GA, Kok RJ, et al. In vitro cellular handling and in vivo targeting of E-selectin-directed immunoconjugates and immunoliposomes used for drug delivery to inflamed endothelium. Pharm Res. 2003;20(1):64–72.
Zingg HH, Laporte SA. The oxytocin receptor. Trends Endocrinol Metab. 2003;14(5):222–227.
Szukiewicz D, Bilska A, Mittal TK, et al. Myometrial contractility influences oxytocin receptor (OXTR) expression in term trophoblast cells obtained from the maternal surface of the human placenta. BMC Pregnancy Childbirth. 2015;15:220.
Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function, and regulation. Physiol Rev. 2001;81(2):629–683.
Takemura M, Kimura T, Nomura S, et al. Expression and localization of human oxytocin receptor mRNA and its protein in chorion and decidua during parturition. J Clin Invest. 1994;93(6):2319–2323.
Anabousi S, Laue M, Lehr C-M, Bakowsky U, Ehrhardt C. Assessing transferrin modification of liposomes by atomic force microscopy and transmission electron microscopy. Eur J Pharm Biopharm. 2005;60(2):295–303.
Bendas G, Krause A, Bakowsky U, Vogel J, Rothe U. Targetability of novel immunoliposomes prepared by a new antibody conjugation technique. Int J Pharm. 1999;181(1):79–93.
Sharkey J, Olcese J. Transcriptional inhibition of oxytocin receptor expression in human myometrial cells by melatonin involves protein kinase C signaling. J Clin Endocrinol Metab. 2007;92(10):4015–4019.
Soloff MS, Jeng YJ, Ilies M, et al. Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector. Mol Hum Reprod. 2004;10(9):685–695.
Maggi M, Peri A, Baldi E, et al. Interferon-alpha downregulates expression of the oxytocin receptor in cultured human myometrial cells. Am J Physiol. 1996;271(5 Pt 1):E840–E846.
Tahara A, Tsukada J, Tomura Y, et al. Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells. Br J Pharmacol. 2000;129(1):131–139.
Busnelli M, Rimoldi V, Viganò P, Persani L, Di Blasio AM, Chini B. Oxytocin-induced cell growth proliferation in human myometrial cells and leiomyomas. Fertil Steril. 2010;94(5):1869–1874.
Molnár M, Rigo J Jr, Romero R, Hertelendy F. Oxytocin activates mitogen-activated protein kinase and up-regulates cyclooxygenase-2 and prostaglandin production in human myometrial cells. Am J Obstet Gynecol. 1999;181(1):42–49.
Ohmichi M, Koike K, Nohara A, et al. Oxytocin stimulates mitogen-activated protein kinase activity in cultured human puerperal uterine myometrial cells. Endocrinology. 1995;136(5):2082–2087.
Phaneuf S, Europe-Finner GN, Varney M, Mackenzie IZ, Watson SP, López Bernal A. Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins. J Endocrinol. 1993;136(3):497–509.
Jeng YJ, Soloff SL, Anderson GD, Soloff MS. Regulation of oxytocin receptor expression in cultured human myometrial cells by fetal bovine serum and lysophospholipids. Endocrinology. 2003;144(1):61–68.
Refuerzo JS, Leonard F, Bulayeva N, et al. Uterus-targeted liposomes for preterm labor management: studies in pregnant mice. Sci Rep. 2016;6:34710.
Vrachnis N, Malamas FM, Sifakis S, Deligeoroglou E, Iliodromiti Z. The oxytocin-oxytocin receptor system and its antagonists as tocolytic agents. Int J Endocrinol. 2011;2011:350546.
Plested CP, Bernal AL. Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium. Exp Physiol. 2001;86(2):303–312.
Phaneuf S, Rodríguez Liñares B, Tambyraja RL, Mackenzie IZ, López Bernal A. Loss of myometrial oxytocin receptors during oxytocin-induced and oxytocin-augmented labour. J Reprod Fertil. 2000;120(1):91–97.
Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG. Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis*. J Biol Chem. 2001;276(22):19452–19460.
Gimpl G, Burger K, Politowska E, Ciarkowski J, Fahrenholz F. Oxytocin receptors and cholesterol: interaction and regulation. Exp Physiol. 2000;85 Spec No:41S–49S.
Gimpl G, Fahrenholz F. Human oxytocin receptors in cholesterol-rich vs cholesterol-poor microdomains of the plasma membrane. Eur J Biochem. 2000;267(9):2483–2497.
Vercauteren D, Vandenbroucke RE, Jones AT, et al. The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. Mol Ther. 2010;18(3):561–569.
Anderson M, Omri A. The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations. Drug Deliv. 2004;11(1):33–39.
Allen TM. Long-circulating (sterically stabilized) liposomes for targeted drug delivery. Trends Pharmacol Sci. 1994;15(7):215–220.
Torchilin VP. Immunoliposomes and PEGylated immunoliposomes: possible use for targeted delivery of imaging agents. Immunomethods. 1994;4(3):244–258.
Norman JE, Bollapragada S, Yuan M, Nelson SM. Inflammatory pathways in the mechanism of parturition. BMC Pregnancy Childbirth. 2007;7(Suppl 1):S7.
Hua S, de Matos MBC, Metselaar JM, Storm G. Current trends and challenges in the clinical translation of nanoparticulate nanomedicines: pathways for translational development and Commercialization. Front Pharmacol. 2018;9:790.
Shojo H, Kaneko Y. Characterization and expression of oxytocin and the oxytocin receptor. Mol Genet Metab. 2000;71(4):552–558.
Bossmar T, Akerlund M, Fantoni G, Szamatowicz J, Melin P, Maggi M. Receptors for and myometrial responses to oxytocin and vasopressin in preterm and term human pregnancy: effects of the oxytocin antagonist atosiban. Am J Obstet Gynecol. 1994;171(6):1634–1642.
Maggi M, Del Carlo P, Fantoni G, et al. Human myometrium during pregnancy contains and responds to V1 vasopressin receptors as well as oxytocin receptors. J Clin Endocrinol Metab. 1990;70(4):1142–1154.
Cassoni P, Sapino A, Fortunati N, Munaron L, Chini B, Bussolati G. Oxytocin inhibits the proliferation of MDA-MB231 human breast-cancer cells via cyclic adenosine monophosphate and protein kinase A. Int J Cancer. 1997;72(2):340–344.
Ito Y, Kobayashi T, Kimura T, et al. Investigation of the oxytocin receptor expression in human breast cancer tissue using newly established monoclonal antibodies. Endocrinology. 1996;137(2):773–779.
Lee KH, Khan-Dawood FS, Dawood MY. Oxytocin receptor and its messenger ribonucleic acid in human leiomyoma and myometrium. Am J Obstet Gynecol. 1998;179(3 Pt 1):620–627.
Cassoni P, Fulcheri E, Carcangiu ML, Stella A, Deaglio S, Bussolati G. Oxytocin receptors in human adenocarcinomas of the endometrium: presence and biological significance. J Pathol. 2000;190(4):470–477.
Cassoni P, Sapino A, Stella A, Fortunati N, Bussolati G. Presence and significance of oxytocin receptors in human neuroblastomas and glial tumors. Int J Cancer. 1998;77(5):695–700.


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Mendel Rosenblum awarded the Inaugural ACM Charles P. Thacker Breakthrough Award
Professor Mendel Rosenblum has been awarded the inaugural ACM Charles P. Thacker Breakthrough Award. The award recognizes individuals or groups with the same out-of-the-box thinking and "can-do" approach to solving the unsolved that Charles Thacker exhibited. Mendel is the DRC Professor in the School of Engineering, Professor of Computer Science, and Professor of Electrical Engineering. He will formally receive the award at ACM's annual Awards Banquet in June, 2019.
Mendel is recognized for reinventing the virtual machine for the modern era and thereby revolutionizing datacenters and enabling modern cloud computing. In the late 1990s, Rosenblum and his students brought virtual machines back to life by using them to solve challenging technical problems in building system software for scalable multiprocessors. In 1998, Rosenblum and colleagues founded VMware. VMware popularized the use of virtual machines as a means of supporting many disparate software environments to share processor resources within a datacenter. This approach ultimately led to the development of modern cloud computing services such as Amazon Web Services, Microsoft Azure, and Google Cloud.
"The new paradigm of cloud computing, in which computing services are delivered over the internet, has been one of the most important developments in the computing industry over the past 20 years," said ACM President Cherri M. Pancake. "Cloud computing has vastly improved the efficiency of systems, reduced costs, and been essential to the operations of businesses at all levels. However, cloud computing, as we know it today, would not be possible without Rosenblum's reinvention of virtual machines. His leadership, both through his early research at Stanford and his founding of VMware, has been indispensable to the rise of datacenters and the preeminence of the cloud."
Please join us in congratulating Mendel for this well-deserved recognition!
Excerpted from "Inaugural ACM Chuck Thacker Breakthrough Award Recognizes Fundamental Contributions that Enabled Cloud Computing", ACM's Latest Awards News, April 10, 2019.
Rosenblum receives DRC Professorship, October 2016
Rosenblum elected to the American Academy of Arts & Sciences, April 2014
Read more about Mendel Rosenblum awarded the Inaugural ACM Charles P. Thacker Breakthrough Award
Dick Pantell
Richard H. Pantell, known widely as Dick, was born in New York City on Christmas Day, 1927. He passed away peacefully in San Mateo, CA on March 26, 2019 at age 91. He is survived by two daughters, Laurie Pantell and Sue Pantell, and his partner Carol Bergman. He was married to Leona Siff Pantell ( BA, 53, LLB 56, Stanford) until her passing in 1996.
Dick got his secondary education at the highly regarded Bronx High School of Science in New York City, graduating in 1944. After a year's study at CCNY, he enrolled at MIT as an Electrical Engineering major. He received both the BSEE and MSEE degrees from MIT in 1950, and his PhD at Stanford in 1954. His PhD supervisor was Professor David Tuttle, a very popular teacher of Network Theory to both graduate and undergraduate students. Professor Tuttle had received his PhD at MIT, studying under the direction of Prof. Ernst Guillemin, himself a network theorist of international reputation.
Dick Pantell's thesis was entitled " New Methods of Driving Point and Transfer Function Synthesis". It proved to be a classic in the field. The earliest work in the general field of passive network synthesis had been done at MIT by Dr. Otto Brune in 1931, who established the necessary and sufficient conditions for a network to be realizable using only resistors, capacitors and inductors. However his realizations sometimes required the use of ideal transformers. The need for ideal transformers was first removed by Dr. S. Darlington at Bell Labs in 1944, but with the constraint that the networks needed to be lossless: composed of only capacitors and inductors.
Dick Pantell's PhD thesis, written in 1954, presented the first general solution to the problem of synthesizing RLC networks with prescribed driving point and transfer characteristics without the limitations required in the Brune and Darlington solutions. It was truly a major contribution to the field. Other work had been done to remove the constraints of Brune and Darlington, but there was no general solution to the problem until Pantell produced one.
Dick was appointed as an Assistant Professor in EE at Stanford in 1956. He proved to be an excellent teacher and supervisor for PhD candidates. He was appointed a Full Professor in 1964.
The breadth of Dick's interests led him to change fields several times, always without fanfare. Following his pathbreaking work in network synthesis, Dick joined the Microwave Laboratory at Stanford to begin working on the design of Megawatt Space-Harmonic Travelling-Wave tubes and related devices. From there, over the next 25 years, he turned sequentially to the study of millimeter wave generation, ferroelectrics, lasers, non-linear optics and photon-electron effects. At the time of his passing, he was working on neutron physics and its application in several areas including materials analysis and medical treatment. His efforts to assist in the commercialization of a small neutron generator for radiology could have important applications in the targeted treatment of specific cancers for decades.
In addition to his scientific interests, he also read widely, bringing a characteristic depth to everything he did, including studying subjects at a sufficient level of detail to write papers and deliver lectures on them. Though he would have bristled at the description, he was what is often called a Renaissance Man.
Read more about Dick Pantell
Information Science & Engineering Night at Elementary School
In March, students enrolled in "EE367A: Information Theory" collaborated with an elementary school in Palo Alto, bringing the younger students interactive games, activities and performance centered around aspects of information theory. More than 50 different activities were were available to the grade schoolers. Students of all ages enjoyed the activities that covered topics such as communication in the animal kingdom; the basics of how DNA carries genetic information; the fundamentals of coding; and even picture books – all geared toward a K-5 audience.
The event was the first of its kind for Professor Tsachy Weissman and his students (pictured). He was inspired by his daughter who sparked the idea by asking to learn more about what he does at his job. The well-attended event welcomed all family members, who kept EE367A students busy answering questions and sharing their own recently acquired knowledge. The collaboration between the two groups will continue into another Science Night to occur later in the next school year.
Thanks to all the terrific students of EE367A!
Pictured above: Irena Fischer-Hwang (project mentor), Yihui Quek (project mentor), Meltem Tolunay (TA), Joachim Neu (project mentor), Tsachy Weissman (professor), Sofia Dudas (student), Logan Spear (student), Shubham Chandak (TA), Ariana Mann (project mentor), and Jay Mardia (project mentor).
All project mentors and course TAs are graduate students currently advised by Professor Tsachy Weissman.
Read more about Information Science & Engineering Night at Elementary School
Prof Jelena Vuckovic named MPQ Distinguished Scholar
Professor Jelena Vuckovic has been named MPQ Distinguished Scholar, 2019. She leads the Stanford's Nanoscale and Quantum Photonics Lab and is Director of Q-FARM (Quantum Fundamentals, Architecture and Machines), a facility of the Stanford-SLAC Quantum Initiative. Jelena is currently a visiting scientist at the Max Planck Institute of Quantum Optics.
The MPQ award recognizes her groundbreaking contributions to the field of Nanoscale and Quantum Photonics. Jelena is the 7th scientist awarded this honor since the Institute was funded. Previous Distinguished Scholars
Please join us in congratulating Jelena on her tremendous research contributions!
About the Max Planck Institute of Quantum Optics
Research concentrates on the interaction of light and matter under extreme conditions. One focus is the high-precision spectroscopy of hydrogen. In the course of these measurements Prof. Theodor W. Hänsch developed the frequency comb technique for which he was awarded the Nobel Prize for Physics in 2005. Other experiments aim at capturing single atoms and photons and letting them interact in a controlled way, thus paving the way towards future quantum computers. Theorists on the other hand are working on strategies to communicate quantum information in a most efficient way. They develop algorithms that allow the safe encryption of secret information. MPQ scientists also investigate the bizarre properties quantum-mechanical many-body systems can take on at extremely low temperatures (about one millionth Kelvin above zero). Finally light flashes with the incredibly short duration of several attoseconds (1 as is a billionth of a billionth of a second) are generated which make it possible, for example, to observe quantum-mechanical processes in atoms such as the 'tunnelling' of electrons or atomic transitions in real time.
Excerpted from "Jelena Vučković named MPQ Distinguished Scholar", Max Planck Institute of Quantum Optics, March 28, 2019.
Read more about Prof Jelena Vuckovic named MPQ Distinguished Scholar
Human compressed images superior to machine-compressed images
The project resulted from a collaboration between researchers led by Professor Tsachy Weissman, and three high school students who interned in his lab.
The researchers asked people to compare images produced by a traditional compression algorithm that shrink huge images into pixilated blurs to those created by humans in data-restricted conditions – text-only communication, which could include links to public images. In many cases, the products of human-powered image sharing proved more satisfactory than the algorithm's work. The researchers will present their work at the 2019 Data Compression Conference.
"Almost every image compressor we have today is evaluated using metrics that don't necessarily represent what humans value in an image," said Irena Fischer-Hwang, an EE grad student and co-author of the paper. "It turns out our algorithms have a long way to go and can learn a lot from the way humans share information."
The project resulted from a collaboration between researchers led by Tsachy and three high school students who interned in his lab.
"Honestly, we came into this collaboration aiming to give the students something that wouldn't distract too much from ongoing research," said Weissman. "But they wanted to do more, and that chutzpah led to a paper and a whole new research thrust for the group. This could very well become among the most exciting projects I've ever been involved in."
Weissman stressed the value of the high school students' contribution, even beyond this paper.
"Tens if not hundreds of thousands of human engineering hours went into designing an algorithm that three high schoolers came and kicked its butt," said Weissman. "It's humbling to consider how far we are in our engineering."
Due to the success of this collaboration, Weissman has created a formal summer internship program in his lab for high schoolers. Imagining how an artist or students interested in psychology or neuroscience could contribute to this work, he is particularly keen to bring on students with varied interests and backgrounds.
Lead authors of this paper are Ashutosh Bhown of Palo Alto High School, Soham Mukherjee of Monta Vista High School and Sean Yang of Saint Francis High School. Weissman is also a member of Stanford Bio-X and the Wu Tsai Neurosciences Institute.
This research was funded by the National Science Foundation, the National Institutes of Health, the Stanford Compression Forum and Google.
Excerpted from "Stanford experiment finds humans beat algorithms at image compression", Stanford News, March 25, 2019.
Read more about Human compressed images superior to machine-compressed images
EE Staff Awards, March '19
Electrical Engineering staff recognized for their outstanding effort include Chet Frost, Joe Little, Rachelle Mozeleski, Helen Niu, and Ryan Samarakoon. Each were nominated by peers, faculty and/or students for professionalism that went above and beyond their everyday roles. Gift card recipients continue to make profound and positive impact in EE's everyday work and academic environment.
Nominations may be submitted at any time. There are no restrictions on the persons or groups that you can nominate. Submitters are asked to include a citation of how the group or person went above and beyond. The submitter can choose to remain anonymous. Link to very brief nomination form.
Please join us in congratulating Chet, Joe, Rachelle, Helen and Ryan. Excerpts from their nominations follow.
Chet Frost, Administrative Associate, Electrical Engineering
"Chet is always quick to respond – and always with a sense of humor and kindness."
"He always goes out of his way to help our group meet requirements and deadlines."
Joe Little, Principal Systems Architect, Electrical Engineering
"Joe always has our back; he was able to fix all my issues really quickly."
"He is extremely capable, responsive and efficient."
Rachelle Mozeleski, Web Content Manager, Electrical Engineering
"Rachelle's creativity and extensive work on her projects is amazing."
"As part of the SEES Committee, she helped transform the annual faculty & staff party into an unparalleled event."
Helen Niu, Administrative Associate, Electrical Engineering
"She has found solutions for new practices, and established efficient procedures moving forward."
"Helen is positive, helpful, understanding, and always goes the extra mile."
Ryan Samarakoon, Life Science Research Lab Manager, Neurosurgery
"Ryan's role is critical and has great complexities - he does a phenomenal job and I trust him completely."
"He manages our experiments efficiently and works very hard to make it easy for us."
The Staff Gift Card Bonus Program is sponsored by the School of Engineering. Each year, the EE department receives several gift cards to distribute to staff members who are recognized for going above and beyond their role. Each month, staff are chosen from nominations received from faculty, students, and staff. Past nominations are eligible for future months.
Nominate a deserving staff person or group today! We encourage you to nominate individuals or groups that have made a profound improvement in daily work life. Each recipient receives a $50 Visa card. Nominations can be made at any time.
Read more about EE Staff Awards, March '19
Dr. Ellen Ochoa named Stanford Engineering Hero
Ellen Ochoa: Ochoa earned her master's and doctoral degrees in electrical engineering at Stanford and soon joined NASA as a research engineer in 1988. In 1990, she was selected as an astronaut and became the first Latina in space, flying aboard Space Shuttle Discovery in 1993. She would go on to log almost 1,000 hours on four separate trips to space. Ochoa then became the 11th director of the Johnson Space Center, the second woman director in the center's history and the first Latina to hold the role. Ochoa received the Distinguished Service Medal, NASA's highest award, and the Presidential Distinguished Rank Award. She has no fewer than six schools named for her, including an elementary school, a public charter middle school and a prep academy.
The Engineering Heroes program, which inducts its eighth class in 2019, was established in 2010 to recognize the profound contributions of distinguished alumni and emeritus faculty of Stanford School of Engineering. Past winners include Nobel Prize winners, inventors, writers, teachers and entrepreneurs who have shaped the world as we know it.
Engineering Heroes are considered and chosen by a panel of technology experts, faculty, alumni, students and historians who are charged with evaluating each nominee's impact and selecting those whose works and ideas set them apart.
This year's Stanford Engineering Heroes are:
Barbara Liskov, computer science
Ellen Ochoa, electrical engineering
Walter Vincent, engineer degree
Please join us in congratulating our outstanding alumni!
news.stanford.edu
Read more about Dr. Ellen Ochoa named Stanford Engineering Hero
Jennifer Widom on the Evolution of Computer Science
Over the course of three decades, Jennifer Widom has seen computer science evolve from a relatively niche field to an interdisciplinary field that touches on broad swaths of society and promises solutions to global problems such as health care and sustainability.
On this episode of the Women in Data Science podcast, Widom explains that as computer science has become more broadly applicable, it has moved beyond the mere study of software and hardware. Today, she says it's about how you can use the methods and techniques in the discipline to solve problems in other fields.
Women in Data Science (WiDS) podcast welcomes Jennifer Widom, "Math, Computers, & Music"
Excerpted from Stanford Engineering Research & Ideas, "Jennifer Widom: Computer science grows beyond engineering disciplines", February 19, 2019.
Read more about Jennifer Widom on the Evolution of Computer Science
Paulraj receives the 2018 IEEE RCC Technical Recognition Award
Professor Emeritus Arogyaswami Paulraj has been selected as the recipient of the 2018 IEEE Radio Communications Committee (RCC) Technical Recognition Award. The award was conferred in December at the IEEE Globecom'18 in Abu Dhabi.
His award citation reads, "For seminal contributions to the theory and practice of multiantenna communications systems".
The Radio Communications Committee (RCC) Technical Recognition Award aims to promote radio communications research and development activities in both the academic and industrial community. This award is established as part of the RCC activities in which research and development takes place in areas related to radio communications. The award recognizes members of the IEEE Communications Society (ComSoc) who have made outstanding contributions to the technological advancement of radio communications.
Please join us in congratulating Paulraj for this well-deserved award!
Read more about Paulraj receives the 2018 IEEE RCC Technical Recognition Award
Thomas Kailath receives the 2018 IEEE RCC Technical Recognition Award
Professor Emeritus Thomas Kailath has been selected as the recipient of the 2018 IEEE Radio Communications Committee (RCC) Technical Recognition Award. The award was conferred in December at the IEEE Globecom'18 in Abu Dhabi.
His award citation reads, "for exceptional contributions to research and education in radio communications".
Congratulations to Tom on this well-deserved award!
Read more about Thomas Kailath receives the 2018 IEEE RCC Technical Recognition Award

redpajama_set_name: RedPajamaCommonCrawl


Embark on a day-long exploration of the trendy and historic district of Belém on the mouth of the Tagus River. Admire the beautiful UNESCO-listed sites at the Belém Tower and the Jerónimos Monastery, see the Discoveries monument and learn all about the area's history from your expert guide.
Meet your guide at Praça do Comércio, one of the largest squares in Europe, and make your way toward Belém. Get up close to the impressive limestone structure of the Belém Tower and learn about its history – the tower dates back to the 16th century, when it was built to support the defence system of the country. If you turn left by the riverside, you can see the brilliant, Golden Gate-esque 25th of April Bridge and take a snapshot of it with the towering Christ the King Statue in the background.
Visit the Portuguese Monument to the Discoveries and listen as your guide explains the symbolism of the sculpture, telling you about brave Portuguese maritime conquests from centuries past. Walk from country to country to mark the discoveries on a large marble map on the floor.
Next, head to Jerónimos Monastery. Admire the breathtakingly beautiful, late Gothic Manueline façades and step inside to discover the sacred complex's marvellous interiors. Your tour ends with a chance to pick up tips from your guide on top restaurants and bars, plus directions to the famous factory – open since 1837 – that produces the delicious custardy pastries known as * pastéis de Belém*.
Children up to 13 years old are welcome to join this tour for free. We reserve the right to ask for a valid ID for children joining our tours.
It was such an entertaining and informative tour due entirely to the engaging and electrifying personality of the tour guide,Chico from Sandeman. I recommend thid tour to everyone visiting Lisbon! They could legitimately charge more if they wanted. Its that fun!
The guide never showed up. He had to pay to take a completely different tour.
Expedia messed up BADLY!! I arrived to the Belem tour at 1430 (AS INDICATED ON EXPEDIA'S WEBSITE) on Wednesday July 13th at the Praca do Comercio only to be told that the Belem tour is at the Paco Luis Camoes and it started at 10 am! !! The tour company worked something out where we completed the Alfama walking tour on Wednesday and return Thursday at 1000 at Praca Luis Camoes and receive a four Euro discount The company informed me that Expedia was advised to change the information on their website TWO WEEKS ago and it has yet to he done! Please get your act together so that other people do not have his experience!!


Wat is dit voor site? Wij zoeken de goedkoopste boeken op internet. Highland Furiesvonden we het goedkoopst op Bol.com voor €24.99.	We hebben gezocht in nieuwe en tweedehands boeken. Rechtsonderaan de pagina kan je het boek direct bestellen	via Bol	.com.
As the oldest of the Highland Regiments, The Black Watch has an enviable roster of Battle Honours and a mystique born of repeated service on behalf of King, Queen and country.
On the strength of her acclaimed biography of Field Marshal Earl Wavell, the regimental trustees commissioned Victoria Schofield to write this, the first volume of her magisterial history of the The Black Watch, and have fully cooperated with her as she traces the story of the Regiment from its early 18th-century beginnings through to the eve of the South African War at the end of the 19th-century.
Originating as companies of highland men raised to keep a 'watch' over the Highlands of Scotland, they were formed into a regiment in 1739. Its soldiers would go on to fight with extraordinary bravery and élan in almost every major engagement fought by the British Army during this period, from the American War of Independence, the Peninsular Wars, Waterloo, the Crimea, Indian Mutiny to Egypt and the Sudan.
Drawing on diaries, letters and memoirs, Victoria Schofield skilfully weaves the multiple strands of this story into an epic narrative of a valiant body of officers and men over one-and-a-half centuries. In her sure hands, the story of The Black Watch is no arid recitation of campaigns, dates and battle honours, but is instead a rich and compelling record of the soldier's experience under fire and on campaign. It is also a celebration of the deeds of a regiment that has played a unique role in British history and a vivid insight into the lives of the many remarkable figures who have marched and fought so proudly under its Colours.


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Warm summer nights. - cocoroxic - Swiss Style Blog.
Warm summer nights.. we wait for them three seasons long and once it arrives it flies by ever so quickly. We try our best to enjoy each and every single second of those days which are supposed to be the best of our lives, yet we still feel like it's never enough. Meanwhile FOMO, the fear of missing out knocks on our door quite frequently to raise awareness that we should move, go out and cherish the days on which the sun decides to shine a little longer than usual. We want to spend time with our loved ones as much as we can, yet we also want to spend it all alone to be able to breathe, to be able to live our lives to the fullest and we get conflicted sometimes. I'm a walking contradiction but I have also learned how to balance it all out at some point. I have learned to let go and let life take its own course. It's not easy to trust the flow when you're a control freak and a perfectionist combined but it is not impossible either. My mind was set to please my people around me but who would please me? That was the question and I now know that this was not important as long as you're in peace with your own soul and know your own pace. Last summer has taught me a lot about patience and forgiveness which I am endlessly grateful for and this summer has taught me that when I accept life the way it is, good things will come your way. I'm not religious, I'm not a believer either yet I believe that the vibes I focus on will notice my needs and serve me with all I desire and truly deserve. This summer has started so well.. it has started out with "a kiss" as Mr. Brightside would chant. It felt like a warm summer night, a soft breeze kissing my skin when it passes by, a dream come true, a prayer that has been heard even though I have never attempted to pray. Warm summer nights gift you with an emotion of feeling home, feeling wanted, feeling like you belong. Warm summer nights make you want to hug the world and radiate positivity from deep within because you have finally found your place after all these battles and hardships having been thrown at you these past few months.. but it was all worth it. Every second of being patience with myself and trusting my instincts have shown that great things really do take time like a fine wine that needs a lot of understanding, care and love united.
With this I want to say that no matter in which phase or state you're in, it's only temporary and that your life will be great again. I want you to know that it is okay to take your time, to follow your own pace and not to rush things.. ever. Does your "warm summer night" rather feel like some kind of "summertime sadness"? That's okay too. Own your emotions, feel them. You don't have to understand them but I want you to allow them to be felt because suppressing them will never help and I know what I'm talking about, trust me. I used to be the queen of avoiding emotions, the ice queen, it always seemed like nothing bothered me until I would drown in my own darkness when left alone with myself for a little too long. I want you to add good things in your life whether you believe you deserve them or not, because I truly believe we deserve the best, always. Add little elements of happiness to your life whenever you can and one day when you take a step back and take a look at the bigger picture having been built up after all this time, you'll realise that it does not take big steps and sudden success.. it only takes little by little and your warm summer night can occur and unfold every so beautifully whenever you so bravely allow it to. I'll be proud of you, my warm summer nights.


Starting today in Australia, Google search results will show information for over 900 commonly searched for health conditions. The new health feature will include an outline of the condition, symptoms, diagnosis, and prevalence according to age at the top of search results. For some conditions you'll also see high-quality illustrations from licensed medical illustrators. Once you get this basic info from Google, you should find it easier to do more research on other sites around the web, or know what questions to ask your doctor.
Think of the last time you searched on Google for health information. Maybe you heard a news story about gluten-free diets and Googled, "What is celiac disease?." Maybe you were planning a trip to Bali, so you looked up "Zika virus" so you could take precaution. Or perhaps you were worried about a loved one, and searched to better understand their condition and prognosis.


"Puraana (the chronicles of the ancients), itivrtta (history), akhyayika (tales), udahaarana (illustrative stories), dharmashastra (the canon of righteous conduct), and arthashastra (the science of government) (- and economics) are known as itihaasa (history)."
Kautilya's Arthashastra -- (Book 1, Ch. 5).
History (Itihaasa) in this definition takes on the meaning more akin to the sense of historiography and is perhaps more eclectic & appears to indicate a super set of political science and history as we use them today.
In the Mahabharata, which is itself considered itihaasa, Adi Parva 1.267-268 mentions that a knowledge of the Itihaasa and Purana is essential for the proper understanding of the veda. By the time the Brahmana and the Upanishada were written, & certainly by the time the Itihaasa and the Purana were written, there was such a well defined sense of history, that the Mahabharata cautions us that the Veda are afraid of those who would read it without a prior acquaintance of the Itihaasa and Purana.
Kalhana in the Rajatarngini states that: "History will be the narration of events as they happened, in the form of a story, which will be an advice to the reader to be followed in life, to gain the purushaarthas namely kama, the satiation of desires through artha, the tool, by following the path of dharma, the human code of conduct to gain moksha or liberation.""
Interesting thoughts especially Kalhana in the Rajatarngini's views on how history links with dharma, artha and moksha.
Could you elaborate on,"..that the Veda are afraid of those who would read it without a prior acquaintance of the Itihaasa and Purana."
I doubt that you have subscribed to the comments section of this post, but if you have, I have this for you.
If you can get your hands on "in search of the cradle of civilization" by Subhash Kak, there, on page 105, 1st paragraph, there is mention of the original Purana which is mentioned in the Vedic literature, that came much before the current Puranas that we know. Perhaps, the Mahabharata was talking of that Purana.


submit receipts to treasurer box in office copy room, 3rd box from the left, top row or scan and email receipts/reimbursement form.
Money/Safe – please arrange with the treasurer or leave money (cash) in the safe. Please make sure the treasurer knows you left cash in safe so they can pick it up.


I bought this item after I ran out of the one I previously used. This was the first time I tried it. I apply smoothly; however, it is really hard to come off since it is waterproof. Great product for the price.
This is a favorite Mascara! It's very popular in my area, so it's extremely hard to find them (they fly off the shelves!).
This mascara is seriously amazing. It gives me full, long lashes that aren't crunchy or spidery. After using this mascara, my lashes look exactly how I want them to look: dark, long, and lush... but still like they could be my natural lashes. However, this mascara loses points in my book because it doesn't hold a curl. I'm constantly on the hunt for a regular (non-waterproof) mascara that keeps my hard-to-curl lashes reaching towards the sky. This mascara didn't do this. If you have hard-to-curl lashes, I suggest choosing the waterproof version, or skipping this mascara altogether. However, if you're blessed with lifted lashes naturally, this mascara might be for you. One of the best parts about this mascara is that it's extremely easy to remove. It will come off effortlessly with any makeup remover or your everyday cleanser. The bottom line: This mascara will make your lashes dark, full, and long; it will not give you fake, spidery lashes. It's also easy to remove, however, it doesn't hold a curl well.
This mascara really isn't worth all the hassle with removing it.
This mascara is very versatile which is definitely something that might interest you. The thing that I like about it is that if I want my eyelashes to be bigger or thicker, I just apply a few more coats and then it works very well for me. It says it's washable, but when I run water over my lashes, it starts to get into my eyes and it really burns. This just seems really cheap and there are much better products out there. Maybelline Full 'N Soft Washable Mascara didn't really work too well for me.
This mascara is perfect just for simple definition of your lashes, especially if you already have long and/or abundant lashes. The selling point of this particular mascara from maybelline is that it will always remain soft even once dry. You have to use make-up remover to get it off because it's waterproof. So laugh til you cry with this on and your lashes will remain beautiful. Performance It works well for what it is intended for.
I used to use this mascara religiously, until I realized I was missing out on mascaras that could give me that super thick, false lash look. However, I think this mascara is the best for sensitive eyes, and also for people who like a natural look. I used to be a contact lens wearer, and now I am a post-LASIK patient. I found no irritation from this mascara. The formula is smooth, not goopy, and it applies light amounts of mascara to the lashes. The brush is fat, so it's going to thicken rather than lengthen. The light formula makes for a soft effect, and if you touch your lashes after, they don't feel crunchy, which defeats the purpose of having pretty lashes to flutter. While I don't reach for this mascara as much anymore because I want a different look, I really think this mascara excels in providing a natural look. This is also great for girls just starting out in makeup because it's not so dramatic.
This is my favorite mascara. I have been using it regularly off and on for many years. I try others but always end up coming back to Full and Soft. This product is inexpensive and works better than more expensive ones that I have tried that are out there on the market. I am wearing it right now and I can run my fingers back and forth over my eyelashes and they feel soft to the touch and they flow easily. Performance It makes my lashes long and full. It spreads my lashes out nicely-they never clump together. My eyelashes are never dry or hard. It never streaks. I could step out into a rainstorm for a moment and not have to worry about mascara lines coming from my eyes. My eyelashes feel soft and healthy. It lasts long and I have never had to reapply during the day. Ease of Application I have never had a problem applying it. The applicator and the bottle are well made. I find less stray eyelashes in the bottle than other mascaras because the brush is quality and the product leaves your eyelashes soft instead of brittle. .
This is by far my must have favorite beauty tool. This mascara is very affordable and does a better job in separating your eye lashes and giving me volume and length than any high priced mascara. It separates each eyelash with its soft bristle brush and makes your eyelashes look like glue on eyelashes. The best part about it is it comes in waterproof or non waterproof and they both give results! Performance Great performance in every way! Ease of Application Easy application.
I don't use this product often but it's works.
I don't use this product every day. But I use it on night outs. It kinda sweats off a little bit, but not completely. It gives major volume. It's not water proof. It's doesn't clump. It's brush provides volume but isn't great for lower lashes. Performance This mascara is fair. It's gives volume. Not too good on length. Not too good on lasting long. It does help grow lashes. I use it on night outs for dramatic lashes because it provides volume. Ease of Application I feel like the brush provides alot of volume. But the brush is not great for lower lashes. It just makes a huge smugdy mess.