Supercritical CO2 assisted liposomes formation: Optimization of the lipidic layer for an efficient hydrophilic drug loading

Journal of CO2 Utilization 18 (2017) 181–188

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Supercritical CO2 assisted liposomes formation: Optimization of the lipidic layer for an efficient hydrophilic drug loading P. Trucillo, R. Campardelli* , E. Reverchon Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano SA, Italy1

A R T I C L E I N F O

Article history: Received 10 November 2016 Received in revised form 17 January 2017 Accepted 1 February 2017 Available online xxx Keywords: Liposomes Supercritical fluids Theophylline Cholesterol Phosphatidylethanolamine

A B S T R A C T

Liposomes are natural vesicles generally based on phosphatidylcholine (PC). The optimization of the lipid bilayer composition with the addition of little percentages of natural lipids is still at early stage due to the difficulties experienced by classical liposome formation processes, mainly, in reproducibility and encapsulation efficiency. Supercritical assisted liposome formation (SuperLip) has demonstrated that these limitations can be overcome. Therefore, in this work, this process has been tested to produce liposomes of controlled nanometric diameter and the effect of water solution flow rate on drug encapsulation efficiency was investigated. The addition of cholesterol (Chol) or phosphatidylethanolamine (PE) was also studied to gain the control on the release rate of the drug entrapped in liposomes. Theophylline was selected as the model hydrophilic drug. Using SuperLip process, PC/Chol and PC/PE liposomes were successfully produced with nanometric mean diameters down to 200 nm. Optimization of both lipid composition and SuperLip operative parameters allowed to obtain theophylline encapsulation efficiencies up to 98%. Drug release kinetics were affected by liposome composition, in particular, the addiction of Chol and PE allowed to slow down theophylline release rate. These results confirmed the possibility of producing liposomes with a complex architecture of the lipid membrane using the SuperLip process. © 2017 Published by Elsevier Ltd.

1. Introduction Liposomes are lipidic vesicles of micrometric or nanometric dimensions, characterized by one or more double layers of phospholipids. Liposome formation process is spontaneous, since phospholipids are able to self-assemble into spherical hollow structures in the presence ofl water, to reach a more favorable energetic state. The closure of the lipidic double layer takes place in successive steps. First lipidic bilayer is planar and flat that corresponds to an unfavorable energetic state. Then the angles of the bilayer plane start to fold like a balloon as a consequence of the interaction with water. Finally, lipidic double layer folds

Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; Chol, cholesterol; MD, mean diameter; SD, standard deviation; PDI, polydispersion index; PSD, particle size distribution; DLS, dynamic light scattering; NTA, nanoparticles tracking analysis; EE, encapsulation efficiency; TE, trapping efficiency; WFR, water flow rate; TEM, transmission electron microscopy; SCF, supercritical fluid; SuperLip, supercritical assisted liposome formation. * Corresponding author. E-mail address: [email protected] (R. Campardelli). 1 www.supercriticalfluidgroup.unisa.it. http://dx.doi.org/10.1016/j.jcou.2017.02.001 2212-9820/© 2017 Published by Elsevier Ltd.

completely, separating the inner water volume by the external one, assuming a spherical shape [1–3]. It is possible to encapsulate either hydrophilic drugs in the inner aqueous compartment, either lipophilic compounds in the lipidic double layer. Lipidic double layer is ideal for drug delivery to the human body, due to the similarity with natural cells [4,5]. Liposomes are not toxic and biodegradable drug delivery systems. Indeed, their lipidic double layer is able to fuse with human cell membrane, becoming part of it (direct cellular up-take) [6]. Vesicles composed of natural phospholipids are also biologically inert if compared to synthetic ones, and this makes them more stable over time [7]. Lipid vesicles are able to preserve drugs from degradative phenomena. Moreover, the controlled release can ensure a constant drug concentration level in the human body. Furthermore, liposomes of nanometric dimensions (below 200 nm [8]) can circulate in the blood stream without being recognized by macrophages; they can easily penetrate into small interstices of tumoral tissues where they can sustain the drug release over days or even weeks [9–11]. In particular, liposomes with nanometric dimensions can avoid sedimentation and blockage of blood circulation [12] and are easily sterilizable being smaller than bacteria average size [13].

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For all these reasons, liposomes are largely studied drug delivery systems to encapsulate, control and protect the release of functional active principles in medical and therapeutic applications, in food, agricultural fields [14] and cosmetic industries [15]. The characteristics of liposomes are strictly related to chemical properties of phospholipids used during the preparation. The kind of lipids selected for liposome formation may modify the total surface charge of liposomes, the permeability, the encapsulation efficiency and the drug release [16–18]. The membrane solidity is often linked to the shape of phospholipids, particularly to the corner amplitude described by their two tails [19]. Phosphatidylcholine (PC) is the most commonly used phospholipid in liposomes membrane [14]. Cholesterol (Chol) is recognized to be compatible with the formation of vesicles. It is reported that it is able to prevent the formation of aggregates and has stabilizing effects. However, a high percentage of cholesterol in liposome formulation can cause liposome destabilization and the presence of Chol crystals in the aqueous bulk. The amount necessary to achieve stable carriers has not been clarified yet [20]. Phosphatidylethanolamine (PE) is another kind of phospholipid used in many cases for liposomes double layer formulations [21]. In this case, the main difference with PC tridimensional structure is the presence of a larger angle between the two lipid chains. PE general shape is truncated conical, while PC has a cylindrical shape [22]. Despite these interesting properties, a systematic study on the effect of PE or Chol incorporation in liposome membranes on vesicles mean diameter, drug encapsulation efficiency, drug release has not been performed yet. For example, some liposome formulations have been developed adding synthetic lipids to PE vesicles, obtaining nanoparticles but with low encapsulation efficiencies of active principles [23]. The effect of cholesterol incorporation was studied together with PEGylation or chitosan coating; but, focusing especially on the permeability effect on the membrane [24]. Methods for the preparation of liposomes have been developed in the last decades [25,26]. However, these methods present some

drawbacks, as low reproducibility, batch operations, low encapsulation efficiency of hydrophilic compounds and a difficult control of liposome size distribution [27]. It is also difficult to remove the solvent from the final suspension, thus hindering the real industrial applications of these proposed technologies [28]. In the field of carrier production, supercritical fluid (SCF) technologies have been proposed to overcome several limitations of conventional processes for the production of micronized particles [29,30] carriers [31–34], coprecipitates [35–41] and nanocomposite polymeric structures [42–44]. Recently, some techniques based on the use of supercritical carbon dioxide (CO2) have been proposed also for liposome production [45–49]. The advantages derived by the use of supercritical carbon dioxide are a larger diffusion coefficient of the lipids and a lower viscosity of the medium bulk. This brings to a better control of PSDs of liposomes produced. However, the supercritical fluids based processes for liposomes production have still some limitations related to the control of liposome dimension and size distribution and also show very low encapsulation efficiency of hydrophilic drug. The major limitation of these processes, both conventional and supercritical, derives from the hydration step of the lipid layer. Indeed, during this step, only a part of the water used for hydration is actually entrapped into liposomes, resulting in a low overall encapsulation efficiency. Recently a supercritical technique, named SuperLip (Supercritical Assisted Liposome Formation) has been developed for the production of liposomes [50]. In this process first water droplets are produced, and then they are rapidly covered by phospholipids. Thanks to the high diffusion coefficient of CO2, the lipids coverage is faster than in the conventional techniques for the production of liposomes. In this way it is possible to produce vesicles with a good control of particle size distribution and high encapsulation efficiency (EE). Indeed, some hydrophilic compounds have been encapsulated inside the aqueous core, with high encapsulation efficiencies up to 97% [51,52].

Fig. 1. Schematic representation of the SuperLip lab-scale plant. R1: CO2 reservoir; R2: lipids reservoir; R3: water solution reservoir; RB: refrigeration bath; P1: CO2 pump; P2: lipids solution pump; P3: water solution pump; S: expanded liquid saturator; PV: precipitation vessel; S: suspension recovery; LS: liquid separator; R: rotameter; DM: dry test meter; TC: thermocouple; M: manometer; MV: micrometric valve; BPV: back pressure valve.

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For these reasons, SuperLip process has been proposed in this work to explore the possibility to produce liposomes with high encapsulation efficiency of hydrophilic compounds and using different lipid mixtures in the bilayer membrane, to understand the effects of lipid composition on drug encapsulation efficiency and drug release kinetics. In particular, the possibility of production of PC based liposomes with Chol and PE in the lipid bilayer (with different w/w percentages) using the SuperLip process is studied, using theophylline as model hydrophilic drug. The effect of water solution flow rate, the presence of Chol and PE is considered on the Particle Size Distribution (PSD), encapsulation efficiency (EE), vesicles stability and drug release kinetics. Liposome stability as well as Chol and PE entrapment efficiency into liposomes bilayer is also investigated. 2. Materials, apparatus and methods 2.1. Materials L-a- phosphatidylcholine from egg yolk (PC,
60% purity, lyophilized powder), L-a- phosphatidylethanolamine from egg yolk (PE, >97% purity, lyophilized powder) and cholesterol powder (Chol, >99% purity, lyophilized powder), were supplied by Sigma Aldrich, Milan, Italy. Ethanol was used as a solvent for lipids and it was purchased by Sigma Aldrich, Milan, Italy (>99.8%). Carbon dioxide was provided by SON, Naples, Italy (>99.4% purity). Distilled water was produced in our laboratories. Theophylline anhydrous powder was purchased by Sigma Aldrich, Milan, Italy (>99%, powder). 2.2. Apparatus The lab-scale plant used for SuperLip experiments has been described in details elsewhere [45]. The complete scheme of SuperLip plant is shown in Fig. 1. Briefly, two different lines were used to feed carbon dioxide and ethanolic solution, containing the lipids, respectively to the same saturator, where an expanded liquid is obtained. The high pressure saturator (internal volume of 0.13 dm3) was filled with stainless steel Berl saddles and thermally heated by thin bands. Then, this supercritical mixture, was fed to a high pressure formation vessel. A 1/8-in. capillary tube fed the expanded liquid to the high pressure vessel (0.5 dm3 internal volume, stainless steel, 25 cm high). A third line was used to deliver the water solution, containing the drug, to the formation vessel, where the atomization of the water solution was obtained using a nozzle of 80 mm diameter. Carbon dioxide was pumped using an Ecoflow pump (mod. LDC-M-2, Lewa, Germany). The head of the pump was continuously refrigerated by a thermostatic bath (mod. FL300, Lauda, Germany), working with a 70:30 (vol/vol) ethylene glycol-water solution. The lipidic solution was prepared with PC and amount of Chol or PE dissolved in ethanol, while the second liquid solution was obtained dissolving theophylline in distilled water. The different solutions were pumped using two separated high pressure precision pumps (Model 305, Gilson, France). Ethanolic solution was fed to the flow rate of 3.5 mL/min, corresponding to 2.76 g/min. Carbon dioxide was fed to the saturator together with ethanolic solution with a fixed Gas to Liquid Ratio (GLR = 2.4, mass based). Operating pressure and temperature were set at 100 bar and 40  C. Different water flow rates were used in this work: 0.7 mL/min, 2.14 mL/min, 10 mL/min. Liposome suspension was recovered from the bottom of the vessel; a decompression step was used to separate carbon dioxide and ethanol using a stainless steel separator. Carbon dioxide flow rate was measured trough a

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rotameter ASA (mod. N.5-2500, Serval 115022) at the exit of the separator. 2.3. Liposome characterizations Liposome suspensions were characterized by Dynamic Light Scattering (DLS) instrument (Mod. Zetasizer Nano S, Worcestershire, UK), to measure mean size (MD), polydispersion index (PDI), standard deviation (SD) and surface zeta potential of the vesicles. This instrument works at 25  C and is equipped with a 5.0 mW HeNe laser operating at 633 nm with a scattering angle of 173 [47]. Transmission electron microscopy, TEM, (JEOL 1400, 100 kV accelerating voltage) was used with negative staining to investigate the morphology and size of the liposomes produced. For samples preparation, a droplet of liposome suspension was placed on a copper grid allowed to sit for 60 s. The droplet was then dried with filter paper. A droplet of staining agent was subsequently placed on top of the grid and left reacting for 30 s, the excess was then removed with filter paper. Nanoparticles tracking analysis, NTA, measurements were performed to determine liposomes concentration in the produced suspensions, using a NanoSight LM20 instrument (NanoSight, Amesbury, U.K.), equipped with a sample vessel and a 640 nm laser. The samples were injected in the vessel using sterile syringes (BD Discardit II) until the liquid reached the tip of the nozzle. All measurements were performed at room temperature. Data were analyzed using NTA 2.0 Build 127 software. The samples were measured for 40 s with manual shutter and gain adjustments. Three measurements of the same sample were performed and values reported are the mean over three measurements. The determination of Encapsulation Efficiency (EE) of liposomes was measured using the supernantant method [48]. In order to obtain an accurate estimation of the drug entrapped, liposome suspensions were ultracentrifugated at 13000 rpm for 30 min at 4  C. Then the amount of theophylline in the supernatant was measured using a Micro-volume UV–vis spectrophotometer (BioSpec-nano, Shimadzu Scientific Instruments, Columbia, USA) at the wave length of 275 nm, as reported in literature [49]. The Encapsulation Efficiency (EE) was calculated as the complement to 100 of the percentage of drug present in the supernatant, as written in the following equation:   ppm ½% EE ¼ 100  1  ppmsupern loaded Encapsulation efficiency tests were performed in triplicates and mean values are reported in this work. Stability tests of liposomes were performed over 5 weeks, measuring the EE during the time of observation. The suspensions were stored at 4  C. Theophylline drug release test at 37  C were studied using UV– vis spectrophotometer. The drug profiles were determined in 80 mL of distilled water continuously stirred at 100 rpm. For each study, 5 mL of theophylline loaded liposome suspension were charged in a dialysis sack (MWCO 3500 Da, Sigma Aldrich, Milan, Italy). Every 30 min the absorbance of theophylline in the aqueous external bulk was measured, drug release tests were performed in triplicates and the curves proposed in the results are the mean profile obtained. The amount of Chol entrapped in the lipidic double layer was measured with Gas Chromatography (GC) assay (GC-FID, mod. 6890 Agilent Series, Agilent Technologies Inc), according to the method reported in the literature [53]. For sample preparation a defined volume of liposome suspension was centrifugated for 50 min at 4  C, then the pellet was re-suspended in 3 mL of hexane and kindly agitated for 30 min. Finally 2 mL of this solution were used for GC analysis.

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The determination of PE amount entrapped in the lipid barrier was not possible to perform, because PC and PE were characterized by very similar GC spectra. 3. Results 3.1. Production of PC/Chol liposomes The basic idea of SuperLip is to modify the steps of liposome preparation. Indeed, in the conventional methods, first a thin lipid layer is produced and, then, liposomes are formed by its hydration. In SuperLip, instead, droplets are first created by the atomization of a water solution; then, atomized droplets are covered by phospholipids in the formation vessel where SC-CO2 and an ethanolic solution containing phospholipids is also flowing. Since the overall scopes of this work are to improve drug release and to have large encapsulation efficiencies, a first set of experiments was performed at different water solution flow rate to verify the possibility of encapsulation of theophylline in PC/Chol based liposomes. The experiments were performed at a pressure of 100 bar and a temperature of 40  C. The flow rate of ethanol solution was set to 3.5 mL/min and carbon dioxide flow rate was calculated to obtain a gas to liquid ratio by mass weight (GLR) of about 2.4. Water flow rate was initially set at 10 mL/min. The corresponding composition of the system during the experiments can be represented on the ternary vapor liquid equilibrium diagram water-CO2-ethanol (neglecting the presence of the solutes), as reported in Fig. 2. In order to obtain liposomes, the operative point should be located inside the miscibility hole, where immiscibility between water droplets and the expanded liquid can be obtained. Outside the miscibility hole, water can be extracted by the expanded liquid solution ethanol-CO2. PC and Chol were dissolved in the ethanol solution (100 mL total volume) to obtain a total lipid concentration in the ethanol solution of 5 mg/mL. Cholesterol was loaded at 1% and 2.5% by weight, with respect to PC amount. PC only based liposomes were also produced for comparison purpose. Theophylline theoretical loading was fixed at 1% w/w with respect to the lipid content. The results are summarized in Table 1. As shown in Table 1, the addition of Chol in the membrane forming liposomes does not affect significantly liposome formation; indeed, the suspensions were successfully produced at all value of Chol concentrations. Size distribution of liposomes produced at different Chol percentages are reported in Fig. 3. Comparing the PSD of PC based liposomes and PC/Chol liposomes,

Fig. 2. Operating points (in gray) in the experiments performed at different water flow rates, reported in the carbon dioxide-water-ethanol ternary diagram at 40  C and 100 bar, adapted from [46].

Table 1 Particles size distribution data for 1% theophylline loaded liposomes with 0%, 1% and 2.5% cholesterol content in the lipid layer, produced at three different water flow rates 10, 2.14 mL/min and 0.7 mL/min respectively. MD: mean diameter, SD: standard deviation, PDI: polydispersion index, WFR: Water Flow Rate, EE: encapsulation efficiency, TE: trapping efficiency. Chol [%]

0 1 2.5 0 1 2.5 0 1 2.5

1% [w/w] theophylline theoretical loading WFR [mL/min]

MD  SD [nm]

PDI

Drug EE [%]

Chol TE [%]

10.0 10.0 10.0 2.14 2.14 2.14 0.70 0.70 0.70

192.2  32 240.5  40 218.7  55 165.9  60 180.1  70 189.8  81 140.5  54 136.2  86 170.5  59

0.171 0.170 0.252 0.362 0.390 0.426 0.470 0.636 0.693

2.0 7.3 5.5 59.5 59.9 58.8 96.3 98.0 97.7

– 25.9 23.7 – 75.8 76.2 – 80.5 81.7

14 PC liposomes Chol 1% liposomes Chol 2.5 % liposomes

12 10 Intensity, %

184

8 6 4 2 0

0

200

400 600 800 Diameter, nm

1000

Fig. 3. 1% w/w theophylline loaded liposomes produced with different cholesterol percentages in the double lipidic layer (0, 1 and 2.5% w/w with respect to the PC mass concentration).

reported in Fig. 3, it is possible to observe that the presence of Chol in the lipid membrane composition did not produce significant effect on vesicles mean diameters and PSDs. In particular, liposome mean diameter values were in the range between 192.2  32 nm and 240.5  40 nm. Nanometric liposomes were produced for all cholesterol concentrations with PDI < 0.2. The experiment with 2.5% of Chol in the lipid layer presented the highest PDI, of about 0.252, with a longer tail. A higher amount of Chol in the lipid bilayer probably causes an enlargement of liposome particle size distribution due to the higher steric volume of chol crystals in the lipid membrane. However, theophylline encapsulation efficiency was very low in all these experiments (see data reported in Table 1). Theophylline encapsulation efficiency seemed to be not affected by Chol percentage in the lipid composition. Also the effective Chol loading inside the lipid membrane, measured using GC analysis, was low with an overall entrapment efficiency of Chol in the bilayer of about 25%. Also the increase of Chol theoretical loading in the initial solution did not largely modify Chol entrapment efficiency. Therefore, some experiments where performed at different water solution flow rates, according to the hypothesis that low encapsulation efficiencies were probably caused by the high velocity of the droplets atomized at the exit of the nozzle and by the disruption of the droplets when they impacted on the water pool located at the bottom of the formation vessel. If these events are considered, the loss of theophylline content from the water internal core of liposomes and also a disaggregation of the lipid bilayer could be explained.

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In order to improve theophylline encapsulation efficiency and to verify the hypothesis of the negative effect of high water flow rate on liposome efficient production, water flow rate was reduced. The same set of experiments with different Chol loading from 0 to 2.5% was repeated at lower water flow rates of 2.14 and 0.7 mL/min. Theophylline theoretical loading with respect to lipid content was maintained at 1% w/w. Results are shown in Table 1. An improvement of both Chol entrapment in the lipid layer and theophylline encapsulation efficiency in the inner water core was successfully obtained operating with a water flow rate of 2.14 mL/ min. Indeed, Chol was entrapped in liposome membrane with a trapping efficiency of about 76.2% and theophylline encapsulation efficiency was improved up to 59.9%. At reduced water flow rate, the phenomenon of disruption of water droplets during the impact with the receiving water bath, at the bottom of the formation vessel, is probably reduced. This allows obtaining higher theophylline encapsulation efficiency and also avoiding the loss of part of the lipid content from liposome membrane. Reducing the water flow rate to 2.14 mL/min, the mean diameter of liposome suspensions was also slightly reduced. In particular, liposome mean diameter values were in the range between 165.9  60 nm and 189.8  81 nm. However, an increase of PSD amplitude was observed, PDI in the range between 0.362 and 0.426 were obtained. The reduction of water flow rate produced a spray of water droplets characterized by a larger size distribution. Comparing the data of PC based liposomes and PC/Chol liposomes, it can be observed that also in this case the presence of Chol in the lipid bilayer composition did not produce significant effect on vesicle mean diameters, but, as also observed in the set of experiments at 10 mL/ min of water flow rate, the wider PSD was obtained at 2.5% of Chol loading in the lipid layer. Indeed, this test presented the highest PDI of the experiments at 2.14 mL/min, of about 0.426, with a longer tail of the PSD, due to higher steric volume of Chol crystals in liposome lipid membrane. Theophylline and also Chol entrapment efficiency was instead not affected by the amount of Chol in the lipid composition of liposome membrane. A further successful increase of Chol and theophylline entrapment efficiency was observed operating with a water flow rate of 0.7 mL/min. Indeed, theophylline encapsulation efficiency was significantly improved up to 98% and Chol entrapment efficiency was improved up to 81.7%. 0.7 mL/min seems to be the best flow rate for an efficient PC/Chol theophylline loaded liposomes production. Liposomes suspensions were successfully produced obtaining a further reduction of liposome mean diameters with values in the range from 136.2  86 nm and 170.5  79 nm. The reduction of water flow rate produced again an enlargement of the PSDs, with PDI over 0.6, confirming the results previously observed. These results seem to confirm that the reduction of water flow rate from 10 to 0.7 mL/min, allows to reduce droplets disruption at the impact on the final water bath avoiding a loss of the encapsulated drugs. 3.2. Production of PC/PE liposomes Another set of experiments was performed using Phosphatidylethanolammine (PE) rather than Cholesterol in the lipid solution. Chol is a one-tail surfactant; instead, PE is a two-tail surfactant. PC is classified as a cylindrical shaped phospholipid, PE is truncated conical shaped [50].The experiments were performed to verify if the shape of phospholipids may play a significant role in the mean dimensions of the produced vesicles. Furthermore, the composition of the lipid bilayer can affect both drug encapsulation efficiency and release kinetics. For this reason, experiments with PC and 0, 1, 2.5% (w/w) of PE content were performed at the operative conditions previously optimized for PC/Chol

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theophylline loaded liposomes: 100 bar, 40  C and 0.7 mL/min water flow rate. Results are summarized in Table 2. Data reported in Fig. 4 and in Table 2 show that the mean diameter of liposomes is not significantly affected also by the presence of PE in the double layer. A slight decrease of liposome mean diameter is observed increasing the PE percentage. For the particular shape of PE tails, probably a lipidic membrane with a denser packing was formed, resulting in lower vesicle mean dimensions [51]. In this case it was not possible to evaluate PE entrapment efficiency in the lipidic double layer because PC and PE were detected in a single GC peak and it was not possible to separate the contribution of the two compounds. Regarding theophylline, instead, encapsulation efficiency up to 95.1% was obtained using the optimized operative parameters. 3.3. Liposome characteristics and drug release rates Transmission Electron Microscope images of liposomes with 1% (w/w) Chol content in the lipidic layer have been produced and reported as examples in Fig. 5a and b. These images report qualitative information about the morphology of the vesicles produced. In particular, it is possible to verify the spherical shape of the vesicles. The sample shows also a relatively wide population of liposomes. It is also possible to observe the presence of the double layer of liposomes, whose thickness is about 30–40 nm. The concentration of vesicles in the aqueous bulk, obtained by Nanoparticle Tracking Analysis (NTA), was about 28.8*106 liposomes/mL. The stability of vesicles is the capability to retain the drug in the inner core. It was measured through the encapsulation efficiency of the sample of liposomes stored at 4  C for over 40 days and controlled every 7 days. Results are reported in Fig. 6 for the sample containing 0.5% of Chol in the lipid membrane and 1% of theophylline in the inner core, produced at 0.7 mL/min of water flow rate. As shown in Fig. 6, the encapsulation efficiency remains stable over 5 weeks, without significative drug leakages. The same behavior was observed for the sample loaded using PE and for PC based liposomes. In conclusion, the presence of different lipids in the liposome membrane did not alter vesicles stability. In order to understand the effect of lipid composition in liposomes membrane on drug release kinetics, samples produced at 0.7 mL/min water flow rate, theophylline loaded at 1% and with 0, 1 and 2.5% w/w of Chol were used to perform drug release tests at 37  C [53]. Results are reported in Fig. 7. From this figure, liposomes with 0% cholesterol (PC based liposomes) released their total content of theophylline in about 5 h. The 1% Chol loaded liposomes completed the release of the drug in about 9 h; whereas, 2.5% cholesterol loaded vesicles released the total drug content after 40 h (about 2 days). These results show that the presence of small quantities of Chol largely modify the drug release kinetics. This experimental evidence is explainable considering that Chol is reported to modify the microviscosity of the lipid membrane, affecting its fluidity; but, also to improve the integrity and the stability of the Table 2 Particles size distribution data of theophylline loaded liposomes with 0%, 1%, 2.5% of phosphatidylethanolammine (PE) content in the lipid layer, produced with the water flow rate of 0.7 mL/min. MD: mean diameter, SD: standard deviation, PDI: polydispersion index, EE: encapsulation efficiency. PE content [%]

MD  SD [nm]

PDI

Drug EE [%]

0 1 2.5

228.9  40 230.9  66 196.6  40

0.179 0.290 0.202

94.5 95.1 96.2

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14

PC liposomes PE 1% liposomes PE 2.5% liposomes

12

Intensity, %

10 8 6 4 2 0

200

400 600 800 1000 1200 Diameter, nm

Fig. 4. PSD of theophylline loaded liposomes produced with increasing percentages phosphatidylethanolamine (PE) encapsulated in the double lipid layer.

loaded in the lipid bilayer, slower drug release kinetics were obtained. In order to understand the effect of PE on theophylline drug release kinetic, a release test was performed for liposomes loaded with 2.5% w/w of PE. Results are shown in Fig. 8, where also the release kinetics obtained for the sample at 2.5% w/w of Chol and for un-processed theophylline are reported for comparison purposes. The presence of PE again slows down the release kinetics of theophylline, indeed PC/PE liposomes released total drug content after about 10 h; whereas, un-processed theophylline is rapidly dissolved in less than 1.5 h. Comparing this result with PC/Chol drug release kinetic, it is possible to observe that the strongest effect on drug release kinetic decrease was obtained when 2.5% of Chol was loaded in the lipid bilayer. This result can be explained considering that, probably, the addition of Chol contribute to increase the integrity and stability of the lipid membrane more

Fig. 5. Transmission Electron Microscope images of liposome vesicles.

than PE. 180

4. Conclusions

Encapsulaon Efficiency, %

160 140 120 100 80 60 EE [%]

40 20 0

0

5

10 15 20 25 30 35 40 Time, days

Fig. 6. Drug retention measurements obtained as Encapsulation Efficiency over a 40 days period on liposomes loaded with 1% w/w theophylline in the inner core and 0.5% w/w cholesterol in the lipidic layer produced using a water flow rate of 0.7 mL/ min.

vesicles’ membrane [54]. Furthermore, cholesterol is capable to increase the hydrophobicity of the membrane [55], inducing longer time retention of hydrophobic molecules, like theophylline, encapsulated in the inner core. For a further confirmation of the previous results, the same tests were performed for the sample produced at 2.14 mL/min of water flow rate and at different Chol percentages. These experiments confirmed the trend observed; i.e., increasing the Chol amount

In this work SuperLip water solution flow rate was modulated to obtain high encapsulation efficiencies of an hydrophilic drug (theophylline) and trapping efficiency of cholesterol and phosphatidylethanolamine in the liposome membrane. The composition of the lipid layer did not affect significantly liposome size distribution, vesicles stability over time and drug encapsulation efficiency, but demonstrated, even at low concentrations, considerable effects on drug release kinetics. The modifications of the fluidity and the permeability of the lipid bilayer with the addition of different lipids into liposome membrane are relevant to prolong drug release rate from these liposomes. Acknowledgements The authors would like to thank Dr. Rossella Crescitelli for the kind help with Transmission Electron Microscope Analysis of liposome suspensions, and also with Nanoparticle Tracking Analysis technique to measure the PSDs. The authors also thank Dr. Mariarosa Scognamiglio with the kind help with Gas Chromatographic measurements to quantify cholesterol encapsulation efficiency in the double lipidic layer of liposomes.

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100 80 Drug release, %

Chol 2.5%

60

Chol 1%

40

PC

20 0

0

5

10 15 20 25 30 35 40 45 Time, h

Fig. 7. Comparison of drug release kinetics of theophylline liposomes loaded with 0, 1, 2.5% w/w cholesterol (Chol) content in the lipid double layer, produced with 0.7 mL/min water flow rate.

Drug release, %

100 80 Chol 2.5% liposomes PE 2.5% liposomes

60

Not encapsulated Theophylline

40 20 0

0

10

20

30

40

50

60

Time, h Fig. 8. Comparison between not entrapped theophylline dissolution kinetics profiles, and entrapped theophylline in 2.5% w/w cholesterol (Chol) loaded liposomes and 2.5% w/w phosphatidylethanolamine (PE) loaded liposomes, produced at water flow rate of 0.7 mL/min.

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