Drug Delivery to the Lungs 27, 2016 – Wachirun Terakosolphan Et Al

Drug Delivery to the Lungs 27, 2016 – Wachirun Terakosolphan et al.

Does glycerol interact with dipalmitoylphosphatidylcholine membranes?

Wachirun Terakosolphan1, Precious Akhuemokhan1, Richard Harvey2 & Ben Forbes1

1Institute of Pharmaceutical Sciences, King’s College London, London, SE1 9NH, UK 2Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle (Saale), 06099, Germany

Summary

In some solution-based pressurised metered dose inhalers (pMDIs), glycerol is used as a non-volatile excipient in order to adjust the aerosol droplet size into the micron range. After actuation, the volatile components of the pMDI formulation evaporate, leaving the drug and glycerol to form the respirable particles. Although the purpose of glycerol is to modify the aerosol particle size distribution, evidence is emerging that it may affect drug kinetics after the particles deposit on the surface of the respiratory tract. Liposomes or monolayers formed from dipalmitoylphosphatidylcholine (DPPC) can be employed as simple models of the epithelial cell membrane, and are used in many pharmaceutical studies to examine the drug absorption or membrane interaction. DPPC also predominates the composition of lung surfactant which coats the mucosa at the glycerol-containing drug particles deposition site. The purpose of this investigation was to conduct a preliminary screen to determine whether glycerol interacts with biomimetic DPPC models as a precursor to more detailed studies into mechanisms that may explain differences in drug permeability in human airways. A fluorimetric assay using laurdan as a fluorescent probe was designed to measure glycerol-DPPC interactions which alter the lipid phase transition temperature. Increasing the concentration of glycerol solution (0, 1.0, 5.0, 10.0, 20.0, and 30.0% w/w) in the DPPC liposome system produced incremental increases in phase transition temperature from 42.6 to 44.2°C. Thus, it can be inferred that glycerol has the potential to modify the molecular rigidity of membrane components which may influence drug permeability in the respiratory tract.

Introduction

According to the Montreal protocol in 1989, chlorofluorocarbon (CFC) propellants were banned from industrial and household products in over 165 countries throughout the world owing to environmental concerns [1]. As a result, CFCs in pressurised metered dose inhalers (pMDIs) were replaced with the alternative propellants, hydrofluoroalkanes (HFAs), which are more appropriate because they are less ozone depleting and in common with CFCs are non-flammable. Conversion to HFA-based pMDI, required additives to the formulations for a variety of purposes. For example, ethanol or isopropanol are used as co-solvents in order to enhance drug solubility in solution-based formulations due to the inferior solubilizing ability of HFAs [2,3]. Several pMDIs were formulated to contain non-volatile components such as glycerol or polyethylene glycol to bulk out the drug particle after dose emission in order to produce a final aerosol droplet in a micron size range similar to that of CFC-based suspension formulations [2–4]. After actuation, all of volatile ingredients including propellant and co-solvent evaporate and leave the drug and glycerol to form the aerosol particles. Evidence is emerging that glycerol may affect drug kinetics after the particles deposit on the surface of the respiratory tract [5,6].

Drug dissolution and absorption in the respiratory tract occurs after particle deposition on the mucosa of the airway or alveolar region. The respiratory mucosa is coated in surface active agents, forming lung surfactant, which lines the air space [7,8]. To examine the effect of glycerol on physiological barriers to drug uptake, it is relevant to study its effect upon lung surfactant and the epithelial cell membrane. Liposomes or air/water interface lipid monolayers formed from dipalmitoylphosphatidylcholine (DPPC) constitute alluringly simple biomimetic models of the cell membrane and as such have been used in many pharmaceutical studies [9,10]. DPPC is also a major component of lung surfactant [11,12]. Thus, DPPC was selected in this study as a model for investigating the effect of glycerol at the particle deposition site.

The aim of this study was to use a simple method to produce DPPC liposomes with various concentrations of glycerol in solution in order to measure their effect on the lipid phase transition temperature using a fluorimetric assay. This is a preliminary study to establish putative biophysical influences of glycerol on model DPPC systems, as a precursor for more detailed investigations into the molecular mechanisms that may explain differences in drug permeability in human airways. These studies will develop our understanding of biological phenomena observed using in vitro models of the site in respiratory tract on which aerosolised particles from glycerol- containing pMDIs have been deposited.

Materials

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoylphosphatidylcholine; DPPC) was purchased from Avanti Polar Lipids (Birmingham, AL), 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (laurdan) was obtained from Molecular Probes (Eugene, OR), chloroform from Fisher Scientific (UK), and glycerol from Sigma-Aldrich (London, UK). Ultrapure water with 18.0 MΩ·cm residual specific resistance was obtained using an Elgastat Maxima purifier (Elga, UK). All other reagents were obtained from standard sources. Drug Delivery to the Lungs 27, 2016 - Does glycerol interact with dipalmitoylphosphatidylcholine membranes?

Experimental methods

DPPC liposomes formation Liposomes were formed by dissolving 10 mg of DPPC and adding a small amount of the fluorescent probe laurdan in chloroform solution to give a total lipid/laurdan molar ratio between 300:1 and 400:1. For the remaining steps after this point, the samples were protected from light to limit photo bleaching of the laurdan by covering the vials and equipment with aluminium foil. The chloroform was removed by evaporation at room temperature in a vacuum desiccator to obtain thin dry lipid films which deposited on the inside walls of the vials. The dried lipid films were resolvated using ultrapure water and various concentrations of glycerol solution (1.0 – 30.0% w/w) to make the final concentration of DPPC in the dispersion at 4 mg/mL. The liposomes were ultrasonicated using a Soniprep 150 ultrasonic disintegrator (MSE, UK) at an amplitude of 10 microns for 5 minutes.

Fluorimetric measurement Steady-state laurdan fluorescence emission was measured using a Varian Cary Eclipse spectrofluorimeter (Agilent, USA) using a 104F-QS semi-micro dual light path (10 × 4 mm) fluorescence cell (Hellma Analytics, Germany). Fluorescence spectra were recorded over a temperature range from 25°C to 65°C increasing in 5°C increments. The temperature of the sample compartment was adjusted using a water-circulating thermostat- controlled Varian Cary Single-Cell Peltier Accessory (Agilent, USA). Additionally, to get a precise reading, the temperature inside the cuvette was directly measured with a K-thermocouple thermometer HI 935005 (Hanna, USA). Emission intensities of all samples were acquired with an excitation wavelength of 340 nm, adjusted for optimal spectral intensity with an excitation slit width of 10 nm and an emission slit width of 5 nm, and scanned from 360 nm to 560 nm.

Data analysis The generalised polarisation (GP) was calculated using the fluorescence intensities at the maximum emission wavelengths of laurdan exhibited when mixed with lipids in either their gel (Ig) or liquid-crystalline (Ic) phases, in this case 440 and 490 nm are used respectively, according to the following formula.

The generalised polarisation as determined using equation (1) was plotted against temperature. Then, the main phase transition temperatures of each sample were obtained from the interception on the X-axis of the GP versus temperature curves.

Results

The fluorescence emission spectra shown in figure 1 are from for laurdan in DPPC multilamellar vesicles at different temperatures, from 25°C (gel phase) to 65°C (liquid crystalline phase), measured in ultrapure water. During the phospholipid phase transition, the spectra show a systematic shift to longer wavelengths as temperature increases; the laurdan emission spectrum in the liquid crystalline phase (high temperature) shows a decrease in intensity at longer wavelengths with respect to the spectrum observed in the gel phase (low temperature). Accordingly, the fluorescence intensities at the maximum emission wavelength of laurdan in the gel and liquid crystalline phases of the lipids, in this case 440 and 490 nm, respectively, were used for the calculation of the GP using equation (1).

Figure 1. Fluorescence emission spectra for laurdan in dipalmitoylphosphatidylcholine multilamellar vesicles at different temperatures, measured at excitation wavelength 340 nm at temperatures between 25-65°C, measured in ultrapure water. Drug Delivery to the Lungs 27, 2016 – Wachirun Terakosolphan et al.

The temperature dependence of laurdan GP values for DPPC MLVs dispersed in various concentrations of glycerol solutions was determined and presented in Figure 2. As expected, the value of laurdan GP decreased from about 0.5 at temperatures corresponding to gel phase lipid to about -0.35 for the liquid crystalline phase. The melting temperature of the glycerol-free system lies between 40°C and 45°C. In the presence of glycerol, the measurements showed a similarity in GP values in the temperature range at 25°C - 35°C and 45°C - 65°C at all glycerol concentrations. However, an increase in GP values in the glycerol-containing liposomes over the range of the phase transition temperature, approximately at 35°C - 45°C, was detected. This difference in GP measurement, especially at the temperature immediately prior to a steep drop in GP, provides evidence for a glycerol-induced modification of membrane phase behaviour. The estimated phase transition temperatures, which were obtained from the intercept on the X-axis of each curve (the temperature at the GP value = 0), of DPPC MLV in 0%, 1.0%, 5.0%, 10.0%, 20.0%, and 30.0% w/w glycerol solution were 42.6°C, 42.9°C, 43.3°C, 43.6°C, 43.7°C, and 44.2°C, respectively (Figure 3).

Figure 2. Temperature dependence of the effect of glycerol 0-30% w/w on the of laurdan GP values in dipalmitoylphosphatidylcholine multilamellar vesicles.

Figure 3. Phase transition temperature of dipalmitoylphosphatidylcholine multilamellar vesicles containing glycerol 0- 30% w/w. Data were obtained from the temperature at the generalised polarisation value = 0 at each glycerol concentration (Figure 2) and expressed as mean + SD (n=3).

Discussion

The phase transition temperature (Tm) of DPPC liposomes was estimated using a fluorimetric technique. Although this biophysical study could not determine directly what happens in the airway when emitted glycerol- containing drug particles deposit there, the alteration in the Tm demonstrates that a modification of membrane phase properties can be caused by glycerol [13]. The transition temperature, which is a specific parameter of each phospholipid, depends on the length of fatty acyl chain, degree of saturation, charge, and the type of head group species [14]. According to the literature, the typical value reported for calorimetric assessment of pure DPPC vesicles is about 41.5°C [15]. From our observations, the estimated Tm of DPPC alone MLVs is 42.6°C, whereas the presence of glycerol raised the phase transition temperature incrementally from 42.9°C to 44.2°C with increasing glycerol content from 1.0 – 30.0% w/w. Factors which can affect the Tm relate to phospholipid aggregate structure and intermolecular interactions, implying that glycerol interacts at some part(s) of DPPC molecules to interfere with intermolecular forces and thus modify the membrane structure. Drug Delivery to the Lungs 27, 2016 - Does glycerol interact with dipalmitoylphosphatidylcholine membranes?

One hypothesis is that glycerol might induce interdigitation of the DPPC bilayers. The interdigitated bilayer is an unusual arrangement of the hydrocarbon chain of phospholipids which exhibits altered physicochemical properties including, size, shape, surface behaviour and phase transition temperature [16]. In an interdigitated DPPC bilayer, the closer association between the hydrocarbon chains of each leaflet might cause greater rigidity, resulting in an increase of the transition temperature [17]. More detailed studies using a wider range of analytical techniques will be required to test this hypothesis.

Conclusion

The results of this study demonstrate that glycerol, which is currently used as non-volatile agent in some pMDI formulations, interacts with DPPC bilayers. This provides the first evidence towards the hypothesis that the effect of glycerol in the emitted particles from pMDI on drug kinetics observed in vitro may be mediated in part through changes in rigidity of epithelial membranes. We speculate that glycerol may increase rigidity in DPPC liposomes by inducing interdigitation of the bilayers. These observations warrant more extensive studies using a wider spectrum of techniques to investigate the effect of glycerol on DPPC membranes in other aspects, for example, structural interaction, alterations in the physicochemical properties of DPPC membranes, and the effect on DPPC monolayers.

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