Desarrollo de los niños en edad preescolar e importancia de esta etapa

2.2.1 Liposome preparation

Liposomes were prepared using the thin film hydration method, as described previously (Ishida et al. 1999). Liposomes were composed of 20 mM soy PC (L-α- phosphatidylcholine) and 0.6 mM mPEG2000-DSPE (1,2-distearoyl-sn-glycero-3-

phosphoethanolamine-N-[(polyethylene glycol)-2000]) (Avanti Polar Lipids, AL, USA), with the addition of either 5 mM cholesterol (Sigma-Aldrich, MO, USA) to form empty liposomes, or 5 mM 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) (prepared in- house (Vine et al. 2016)) to form N-AI-loaded liposomes. Reagents were weighed out into a round-bottom flask and dissolved in a 2:1 (v/v) mixture of chloroform/methanol (Sigma-Aldrich, MO, USA). Organic solvents were removed by rotary evaporation and subsequent freeze drying to form a lipid film. Dried liposome films were rehydrated in deoxygenated 25 mM HEPES buffer (115 mM NaCl, 20 mM HEPES, 2.4 mM K2PO4,

1.2 mM CaCl2, 1.2 mM MgCl2; pH 7.4) at a phospholipid concentration of 20 mM by

shaking for 1 h at room temperature (RT) with intermittent sonication. Once reconstituted, liposomes were passed through a 0.22 µm polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Germany) and then serially extruded 11 times through a 0.1 µm PVDF membrane using a syringe-driven extruding apparatus (Avanti Polar Lipids, AL, USA) at RT.

2.2.2 PAI-2 conjugation to liposomes

Liposomes were surface-functionalised with PAI-2 using either the conventional (CO) method or post-insertion (PI) method (Allen et al. 2002). Human recombinant PAI-2, ΔCD-loop, prepared as described previously (Cochran et al. 2009), has free cysteine residues for conjugation to terminal maleimide groups of maleimide-functionalised PEG (Oswald et al. 2016). For the CO method, preformed liposomes were incubated with PAI- 2 at a molar ratio of 3333:1 liposome phospholipid:protein for 2 h at RT. For the PI method, micelles composed of 0.8 mM mal-PEG2000-DSPE (1,2-distearoyl-sn-glycero-3-

33 mPEG2000-DSPE were prepared as per previously reported methods (Moreira et al. 2002),

and PAI-2 was added to the micelles at a molar ratio of 10:1 (mal-PEG2000-DSPE:protein)

to form PAI-2-functionalised micelles. PAI-2-functionalised micelles were added to preformed liposomes and heated to 60°C for 1 h to facilitate the post-insertion of micelle lipids into the outer leaflet of the liposomes. Following the liposome functionalisation steps, unbound PAI-2 was removed from liposomes by either size-exclusion chromatography (SEC) using Sepharose CL-4B (Sigma-Aldrich, MO, USA) according to the manufacturer’s protocol, or repeated centrifugation at 20,000 x g for 1.5 h at 4°C.

2.2.3 Liposome characterisation

2.2.3.1 Dynamic light scattering

Liposome size distribution, peak intensity and polydispersity index (PDI), as well as stability over time using repeated measurements, were determined by dynamic light scattering (DLS) using a Zetasizer APS instrument (Malvern Instruments, UK). Liposome samples (60-100 µL) in phosphate-buffered saline (PBS) or HEPES were added to 96- well plates and analysed at 25°C using the manufacturer’s measurement protocol for liposomes (13 reads per sample, triplicate measurements). Data were presented as the intensity distribution for liposomes and the number distribution for micelles in order to enable visualisation of small particles. Zeta potential (surface charge) of liposomes was determined by DLS using a Zetasizer Nano ZS instrument (Malvern Instruments, UK) (10-100 reads per sample, triplicate measurements).

2.2.3.2 Nanoparticle tracking analysis

Liposome size distribution and particle concentration were determined using a NanoSight LM 10 instrument (Malvern Instruments, UK) according to the manufacturer’s protocol. Liposomes were diluted to a concentration of between 1×108 and 25×108 particles/mL in PBS to ensure an optimal concentration for accurate analysis of samples (NanoSight Ltd, UK). Imaging of 20-100 particles per field of view was performed using an optical microscope fitted with a charge-coupled device camera at 25°C. Particle movement was recorded at 20 frames per second for 60 seconds. Average particle size and concentration were calculated using Nanoparticle Tracking Analysis (NTA) software (version 2.3, NanoSight Ltd, UK) from triplicate measurements.

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2.2.3.3 Phospholipid assay

Liposome phospholipid concentration was determined using a commercial phospholipid kit (Sigma-Aldrich, MO, USA), as per the manufacturer’s instructions. Briefly, phosphatidylcholine standards were prepared 0-20 µM, 20 µL of each standard was added to a 96-well plate in duplicate, as well as 20 µL of each liposome sample (at various dilutions). 80 µL of reaction mix containing assay buffer, enzyme mix, phospholipase D enzyme and dye reagent, was added to each well and incubated at RT for 30 min. Absorbance was measured at 570 nm using a Spectramax spectrophotometer (Molecular Devices, CA, USA). The absorbance values of the phospholipid standards were used to create a standard curve and unknown sample concentrations were determined by interpolation.

2.2.3.4 N-AI encapsulation in liposomes

Drug encapsulation efficiency was determined by high-performance liquid chromatography (HPLC). N-AI-loaded liposomes were mixed with water/acetonitrile (60:40 v/v) and centrifuged. The N-AI concentration was determined using an Atlantis T3 reverse-phase C18 analytical column (Waters, UK) and a Waters HPLC machine (Waters, MA, USA). Analysis was performed using an injected volume of 10 µL with a gradient elution and monitored with a photodiode array at 435 nm. Concentration was determined by interpolating from a standard curve after analysis of standards and samples using Empower Pro V2 software (Waters, UK).

2.2.3.5 Lowry assay

Protein concentration of PAI-2 and PAI-2-functionalised liposomes was determined using the DC Protein Assay kit (Bio-Rad Laboratories, CA, USA) according to the manufacturer’s protocol. Bovine serum albumin (BSA) was used as a standard (0-2 mg/mL) and 5 µL of standard or sample was added to a 96-well plate. Bio-Rad reagent A (25 µL) and reagent B (200 µL) were added to each well, the plate incubated at RT for 10 min, and the absorbance measured using a Spectramax spectrophotometer (Molecular Devices, CA, USA) at 750 nm. The absorbance values of the BSA were used to create a standard curve and unknown sample concentrations were determined by interpolation.

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2.2.3.6 BCA assay

The Bicinchoninic Acid (BCA) assay is one of the most sensitive colourimetric protein assays and can detect protein at concentrations as low as 5 µg/mL (Walker 1996). Due to phospholipid interference in the Lowry analysis of liposome samples, protein concentration of PAI-2-functionalised liposomes was additionally determined using the BCA assay according to previously published methods. Solution A, consisting of 20 g/L sodium carbonate, 9.5 g/L sodium bicarbonate and 1.6 g/L sodium tartrate, was brought to pH 11.25 with the addition of 1 M sodium hydroxide. Immediately prior to use, 0.5 g BCA powder (Sigma-Aldrich, MO, USA) was dissolved in 50 mL of solution A. Solution A was combined with solution B, which was made up of 4% (w/v) copper sulfate in water, in a ratio of 50:1 to produce BCA working reagent (solution C). BSA protein standards, ranging in concentration from 0-1 mg/mL, were prepared in PBS, and 10 μL of each protein standard was added to a 96-well plate in triplicate. Various dilutions of samples in PBS were prepared and 10 μL of each solution was added to the plate in triplicate. 80 μL of solution C was added to each well and the plate incubated at 60°C for 15 min. The absorbance of the solutions within the wells was determined using a FLUOstar OPTIMA plate reader (BMG Labtech, Germany) at a wavelength of 544 nm. The absorbance of the BSA was used to create a standard curve and unknown sample concentrations were determined by interpolation.

2.2.3.7 SDS-PAGE

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, loading buffer (containing 50% (v/v) glycerol, 2% or 6% (w/v) sodium dodecyl sulfate and 0.02% (v/v) bromophenol blue in distilled water) was added to samples. For reducing conditions, 5 μL of β-mercaptoethanol (Sigma-Aldrich, MO, USA) was added to samples. Samples were denatured by heating to 100°C for 5 min and were then loaded into a 10% gel to run by SDS-PAGE at 100 V for 2 h.

2.2.3.8 Western blotting

Western blotting was used to detect and quantify PAI-2 conjugated to liposomes. Proteins in SDS-PAGE gels were transferred to PVDF membranes using Bio-Rad transfer equipment (Bio-Rad Laboratories, CA, USA) at 100 V for 1.5 h. Membranes were rinsed in TBST (1X TBS buffer with 0.05% v/v Tween-20) and blocked using 10% skim milk

36 in TBST for 1 h at RT. After rinsing membranes twice with TBST, membranes were incubated with primary antibody (anti-SerpinB2; Abcam, Cambridge, UK) at 1:2000 dilution in 2% skim milk/TBST at 4°C overnight. Membranes were washed with TBST four times (10 min each wash) and then incubated with secondary antibody (anti-rabbit- HRP; Abcam, Cambridge, UK) at 1:5000 dilution in 2% skim milk/TBST for 2 h at RT. Membranes were then washed in TBST three times for 5 min and then in TBS (no Tween- 20) three times for 5 min. Membranes were developed using ECL peroxidase reaction (Pierce PicoWest ECL reagent; Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. Membranes were visualised using x-ray film after developing and fixing (Bio-Rad Laboratories, CA, USA) or using a Gel Logic 2200 Digital Imager (Carestream Molecular Imaging, CT, USA). Band intensities were quantified using ImageJ (National Institutes of Health, MD, USA).

2.2.3.9 Flow cytometric analysis of liposomes

Flow cytometry was used to further assess liposome size and quantify the efficiency of micelle insertion into preformed liposomes. Fluorescent liposomes were prepared by loading with rhodamine-123 (R123) (Sigma-Aldrich, MO, USA) or by post-inserting micelles that were prepared in the absence or presence of varying percentages of FITC- DSPE-PEG (Avanti Polar Lipids, AL, USA). Events (50,000) were collected using an LSR II flow cytometer (BD Biosciences, NJ, USA; excitation 488 nm, emission collected with a 515/20 band-pass filter). Data were analysed using FlowJo software version 10 (FlowJo LLC, OR, USA).

2.2.3.10 Fluorogenic uPA activity assay

To determine whether PAI-2 conjugated to the surface of liposomes retained inhibitory activity against uPA, the activity of PAI-2-functionalised liposomes was quantified using a fluorogenic uPA activity assay, as described previously (Cochran et al. 2009). Briefly, liposomes were diluted in 100 μL reaction buffer (20 mM HEPES, pH 7.6, 100 mM NaCl, 0.5 mM EDTA, 0.01% (v/v) Tween 20) containing 0.25 mM uPA fluorogenic substrate (Z-Gly-Gly-Arg-AMC; Merck Millipore, MA, USA). After a brief pre-incubation at 37 ºC, high molecular weight urokinase plasminogen activator (HMW-uPA) (final concentration 0.675 nM) was added to start the reaction and fluorescence emission was

37 measured at 37 ºC using a microplate reader (POLARstar Omega; BMG Labtech, Germany). All assays were performed in triplicate and values corrected by subtracting the background well values (reaction buffer and substrate only).

2.2.4 Data analysis

All data analysis, including the generation of graphs and statistical tests, was performed using GraphPad Prism version 7 for Windows (GraphPad Software, CA, USA), unless stated otherwise. Data are presented as the mean ± standard deviation (s.d.) or standard error of the mean (s.e.m.) as stated. Pairwise comparisons were made using Student’s t- test and multiple comparisons were made using one-way ANOVA with Tukey’s post- test.