Dispersibility of phospholipids and their optimization for the efficient production of liposomes using supercritical fluid technology

Accepted Manuscript Dispersibility of phospholipids and its optimization for efficient production of liposomes using supercritical fluid technology Faheem Maqbool, Peter M. Moyle, Kristofer J. Thurecht, James R. Falconer PII: DOI: Reference:

S0378-5173(19)30245-5 https://doi.org/10.1016/j.ijpharm.2019.03.053 IJP 18237

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

2 February 2019 23 March 2019 25 March 2019

Please cite this article as: F. Maqbool, P.M. Moyle, K.J. Thurecht, J.R. Falconer, Dispersibility of phospholipids and its optimization for efficient production of liposomes using supercritical fluid technology, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.03.053

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Dispersibility of phospholipids and its optimization for efficient production of liposomes using supercritical fluid technology Faheem Maqbool1, Peter M. Moyle1, *, Kristofer J. Thurecht2, James R. Falconer1, * 1

School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia

2

The Centre for Advanced Imaging (CAI), The University of Queensland, Brisbane, QLD

4072, Australia

*Co-correspondence: [email protected] (J.R.F) Tel: +61(4) 312 73844 and [email protected] (P.M.M.) Tel: +61(7) 334 61869

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Graphical Abstract:

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Abstract 5

Liposomes are promising delivery vehicles and offer the added drawcard of being able to be made functional to target tissues such as cardiac muscle and cancerous cells. Current methods to manufacture liposomes need to be improved and supercritical fluid (SCF) technologies may offer a solution. Herein, the dispersibility of six different phospholipids (PLs): determined using supercritical carbon dioxide (scCO2), and 1,2-distearoyl-sn-glycero-3-

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phosphocholine (DSPC) showed highest post-processing dispersibility, while 1,2-dioleoyl-snglycero-3-phosphocholine

(DOPC),

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,

(DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) showed no dispersibility at all in scCO2 at the assessed experimental conditions. The zetasizer results showed that the SCF conditions at 37 °C, 250 bar and 200 RPM for 60 min provided nanoparticles with 15

narrowest polydispersity index (PDI) and spherical shaped as shown by cryo-transmission electron microscopy (Cryo-TEM) supported these results. The mean diameter of liposomes using the SCF method for DSPC-PEGylated and DOPC-PEGylated liposomes was 98.3±3.3 nm and 124.5±4.1 nm, while using thin film method it was 153.6 ± 4.5 nm and 131.3±3.4 nm, respectively. The stability of liposomes stored at different temperatures (25 °C, 4 °C and

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-20 °C) using SCF technology was better over a period of 3 months. The current study would provide green alternative method, less laborious, save time and energy. Keywords: Green technology, Zwitter-ionic, Electron microscopy, Particle engineering, Drug delivery, Size distribution

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1. Introduction Liposomes are nano/micro-sized spherical shaped vesicles, composed of an inner aqueous layer enclosed by a phospholipids (PLs) bilayer. PLs are the structural building blocks of liposomes that ; (i) are biocompatible (Daraee et al., 2016) (ii) have hydrophilic head groups and hydrophobic tail (Torchilin, 2005) and have a similar structural composition to cell

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membranes in the human body. Liposomes can be unilamellar or multilamellar, based on the number of lipid bilayers; therefore, their size can vary from nano- to micro-meter range (Gortzi et al., 2007; Joshi and Müller, 2009). Liposomes are useful drug delivery system, as they can deliver either hydrophobic or hydrophilic drugs, even possible for both at the same time.

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Liposomes have had commercial success in delivering anticancer drugs at cancerous tissues (Brannon-Peppas and Blanchette, 2012). As of writing, the FDA has approved 15 liposomal formulations on the market, seven are used for cancer therapy (Bulbake et al., 2017). Research on liposomes in recent years has been rapidly growing, and liposomes have progressed from basic delivery systems to immune evaded and targeted systems (Akbarzadeh

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et al., 2013). Liposomes take advantage of tumor leaky microenvironment; thus liposomal nanoparticles (100-400 nm) cross the endothelium gaps with the increased permeation effect and enhanced retention at tumor site due to poor lymphatic drainage. In addition, stealth liposomes possessing polyethylene glycol (PEG) can increase the circulation time of liposomes by reducing immune-mediated clearance of liposomes by the reticoendothelial

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system. The effects of polymers associated with different PLs (e.g. charge, liposome particle size and stability) have shown promising results in targeting different types of cancerous cells (He et al., 2010). Liposome-based drug delivery systems are also desirable due to being nontoxic and biodegradable within the human body (Gregoriadis and Ryman, 1971; Liu and Boyd, 2013; Sharma and Sharma, 1997). 4

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Several methods have been developed to produce liposomes. These include the original Bangham method, also known as the thin film method (Bangham et al., 1965), reverse phase evaporation, ether injection (Deamer and Bangham, 1976; Schieren et al., 1978), freeze thaw (Pick, 1981), detergent depletion (Torchilin and Weissig, 2003), membrane contactor (Charcosset and Fessi, 2005), microfluidics (Jahn et al., 2007), post-

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formation/homogenisation, and emulsion methods (Batzri and Korn, 1973; Deamer and Bangham, 1976). Each method has its own drawbacks, such as the use of large amounts of organic solvents, multiple steps (thus time consuming), environmental waste issues, and stability complications. Supercritical fluid (SCF) technology has the potential to provide an alternative to address these problems. SCF as a solvent method can be utilised with less/no

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use of organic solvent, thus a green/er technology, and operating can use cheap, readily available and non-flammable gas like carbon dioxide (Alnaief and Smirnova, 2011; Prosapio et al., 2015), producing a solvent-free end product. Supercritical carbon dioxide (scCO2) is one of the most commonly used SCFs due to its low critical temperature (Tc) i.e. approx. 32 °C (305 Kelvin) in pure form, which is less

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likely to cause degradation to a thermolabile compound and only requires a small amount of heat (energy), thus adding to the green method banner, its critical pressure (Pc) in pure form is approx. 73 bar (1059 psi) (Esfandiari, 2015; Poliakoff et al., 2014; Zheng et al., 2016). It is interesting to note, that scCO2 methods produce a zero-net change in atmosphere amounts of CO2, as the CO2 used in processing is gathered from industrial sites. In addition, SCF

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technologies can be scaled up, so simpler lab-bench equipment is sufficient for optimising methods that can be extrapolated to much larger sized equipment (Cansell et al., 2003; Cardea et al., 2010; Hakuta et al., 2003; Huang et al., 2005; Kim et al., 2007; Shariati and Peters, 2003; Wu et al., 2015). These desirable attributes create a need to explore new methodologies using scCO2 in the field of liposomal drug delivery and nanotechnology.

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Although, use of acCO2 has been previously reported in many studies for natural product extraction from plants and in the area of polymer chemistry (Ewing and Kazarian, 2018; Lorenzen et al., 2017; Nuchuchua et al., 2017; Stadie et al., 2015; Trucillo et al., 2017), the gap needs to be filled in the field of liposomal drug delivery and nanotechnology. The minimal work has been done on the dispersibility of different charged groups of PLs in

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scCO2 and production of liposomes using these PLs and SCF technology. Although some studies using other techniques have been published before but all of these studies have used anti-solvents techniques and other PLs. Thus, there is need to provide novel data for manufacturing liposomes using six PLs and SCF technology. In this study, SCF has been used as main solvent and co-solvent has been used to improved dispersibility of few PLs.

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The aim of this study was to determine the dispersibility of six different charged PLs in scCO2 to optimize the production of liposomes using a SCF method. The PLs were selected based on those used in the market and classed as either having a +ve or -ve charged head group or zwitterionic, viz having opposite dual charges. There were six PLs selected; a. DSPC, b. DOPC, c. 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), d.

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1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), e. DOTAP, f. DOPE. (See Table 1). In addition, the PEGylation of these PLs was evaluated for scCO2 dispersibility and to produce stealth liposomes. These were DSPC-PEG and DOPC-PEG liposomes, which were produced with the similar molar ratio using SCF technology, as available in the market. The scCO2 conditions were screened for effects on PL dispersibility and included;

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varying temperature, pressure, time, and their effects were evaluated on the particle size, PDI and liposome morphology. Moreover, liposomes produced using SCF technology were compared to the traditional thin film method. Moreover, for stability studies, the liposomes stored at different temperatures (25 °C, 4 °C and -20 °C) were analysed in terms of change in the PDI and particle size for the period of 3 months.

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2. Materials and methods 2.1. Materials DSPC, 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2-dioleoyl-snglycero-3-phospho-(1'-rac-glycerol) (DOPG), DOPE, DOPC, DOTAP and 1,2-distearoyl-snglycero-3-phospho- ethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)

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(DSPE-PEG-2000) were purchased from Avanti Polar Lipids, Alabama, USA. Ethanol and carbon dioxide liquid cylinder were obtained from Merck Millipore (Kilsynth, VIC, Australia) and BOC Australia, respectively. All solvents used in the study were of analytical or high-pressure liquid chromatography (HPLC) grade. 2.2. General method of processing phospholipids using SCF technology.

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In this section, the general method to process PLs using scCO2, for determination of dispersibility and manufacture liposome has been described in detail. The modifications of different parameters including optimization for screening the best processing conditions is explained in the later sections. The stainless steel (SS) vessel (360 grade with 60 mL 115

capacity) was used as reaction/processing vessel for the experiment. At first, the SS vessel was sealed, and liquid CO2 was injected by a syringe pump and converted into scCO2. The CO2 becomes scCO2 above Tc and Pc and for each experiment, a specific temperature and pressure was used, as explained in the following sections in detail. It took 2-3 min to fill the CO2 into SS vessel when steady state was achieved. The overhead stirrer fitted above the SS

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vessel used to rotate the peddle (hanging in the high-pressure SS vessel) for mixing and provide agitation and the temperature of the system was controlled by the heating jacket and monitored using an inlet probe passing through the SS vessel (see Figure 1, schematic diagram for experimental set-up). At the end of experiment, the vessel was slowly

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depressurized to atmospheric conditions and sudden drop of pressure led to the formation of 125

CO2 gas from scCO2, which was then released back into the air. 2.3. Determination of dispersibility of phospholipids using subcritical and supercritical carbon dioxide To determine dispersibility of all PLs, based on the different head groups charge; DSPC, DPPG, DOPG, DOPE, DOPC and DOTAP were processed using liquid (subcritical) and

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scCO2 in separate experiments. For all experiments 20 ± 0.1 mg of the PLs were used in individual set of experiment. The processing conditions were set at 37 °C and 60 bar (for liquid/subcritical CO2) and 37 °C with 250 bar (for scCO2) with 200 RPM, in a 60 mL highpressure SS vessel for 60 min. The steps of this experiment were performed in similar order, as explained above in the general method section. At the end of experiment, to

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assess/determine the dispersibility of PLs in scCO2 bird-eye images of the inner section/lining of SS vessel were collected before processing (0 min) and after depressurization.

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Figure 1: Schematic diagram of the experimental setup to determine the dispersibility of phospholipids (PLs) for production of liposomes using scCO2 in a high-pressure stainless steel (SS) vessel.

2.4. Manufacturing conventional liposomes using SCF technology The SCF technology was first used in the current study to manufacture conventional/nonstealth liposomes of the all PLs (See Table 1). To manufacture 5 mM (94: 6 molar ratios 145

(PLs: DSPE-PEG-2000) of conventional liposomes of six PLs, the experiments were performed at 37 °C, 250 bar, 200 RPM in 60 mL of scCO2. After completion of experiment, the thin film of proliposomes was formed, which was processed for hydration to manufacture liposomes using 5 mL of normal saline for 30 min above transition temperature (TT) (60 ˚C for DSPC and 50 ˚C for all other PLs). To perform hydration of the thin film of 9

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proliposomes, 5 mL of the normal saline was added into the high pressure stainless steel vessel and placed on the hot plate to perform hydration and stirring (100 RPM using magnetic stirrer/hot plate, at temperature above TT of respective PLs) to manufacture liposomes. 2.4.1. Asses the effect of transition temperatures

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To see the effect of temperatures above TT of the PLs, additional experiments were performed for DSPC (at 56 °C) and DPPG (at 42 °C) (see Table 1 for TT of PLs). The particle size analysis of the liposomes was performed and compared with the liposomes manufactured using temperature below TT of PLs. 2.4.2. Effect of co-solvent

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To assess the effect of co-solvents with scCO2 on the dispersibility of PLs, such as: DOPC, DOPE and DOTAP; 0.5 mL (~ 0.74 % of the total mass ratio of PLDs and DSPE-PEG-2000) of ethanol was used with 60 mL of scCO2. To do this, first PLs were dissolved in ethanol and then the solution was added into the high-pressure SS vessel. The similar SCF general method was used, as explained in above section at 37 °C, 250 bar, 200 RPM. After

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depressurized of the of SS vessel proliposomes were hydrated using 5 mL of normal saline above TT of the PLs for 30 min. 2.4.3. Effect of sonication To see the effect of sonication on particle, size, population distribution and morphology of the manufactured liposomes, sonication was performed using Grant XUB18 Ultrasonic Water

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Bath for 15 min. To analyse these effects the particle size analysis was performed of the manufactured liposomes before and after sonication and compared accordingly.

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Table 1. Transition temperatures and surface charges of the selected phospholipids (PLs) used in the study. Type of PLs DSPC DOPE DOPC DOTAP DPPG DOPG

Charge on PLs Transition Temperature (°C)

Zwitterionic

Zwitterionic

Zwitterionic

Cationic

Anionic

Anionic

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-16

-17

5

41

-18

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2.5. Manufacturing PEGylated liposomes using SCF technology To manufacture PEGylated liposomes of DSPC and DOPC using SCF technology, same method was used, as described in the above section. The conditions were 37°C, 250 bar, 200 RPM in 60 m L of scCO2. The initial data of dispersibility and conventional liposomes using 180

scCO2, provided further direction to manufacture PEGylated liposomes. The composition of PLs and DSPE-PEG used to manufacture liposomes was like the market available stealth liposomes (DOXIL®). DSPC with DSPE-PEG-2000 (94: 6 molar ratio) and DOPC with DSPE-PEG-2000 (94: 6 molar ratio) were dispersed into 60 mL of scCO2 (SS vessel) and processed at various conditions detailed in the following sections (Naik et al., 2010). For

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DOPC PL, 0.5 mL of ethanol was used as co-solvent with 60 mL of scCO2. The unsaturated PL e.g. DOPC show maximum drug loading capacity for hydrophobic drugs compared to the saturated PL. The basic reason of this high loading is associated with wide lipid bilayer, compared to the saturated PL (Hong et al., 2015). To do this, first DOPC was dissolved in ethanol and the solution added into high-pressure SS vessel. The effect of different

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modifications was studied and is explained in the following sections. 2.5.1. Effect of varying temperature and pressure To see the effect of varying temperature, pressure on the hydrodynamic particle size and PDI of PEGylated liposomes, number of experiments were performed with different combinations of temperature and pressure. The conditions used were 100, 150, 250 and 300 bar processed

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at constant temperature i.e. 37 °C in separate experiments. Moreover, DSPC-PEGylated 11

liposomes were also prepared at 56 °C (above TT) and 250 bar. The particle sizing data and PDI of the liposomes was taken after each experiment and compared accordingly. 2.5.2. Effect of time and sonication To see the effect time (experiment time) on particle, size, distribution and morphology, 200

separate experiments were performed at 30 and 60 min with similar pressure and temperature combinations, as explained above. In addition, effect of sonication was evaluated using Grant XUB18 Ultrasonic Water Bath for 15 min. The sonication was performed after hydration and manufacturing of PEGylated liposomes. In addition, particle size and PDI of the PEGylated liposomes was determined before and after sonication and compared accordingly.

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2.6. Manufacturing PEGylated liposomes using thin film (Bangham) method To prepare (5 mM of liposomes with 94: 6 molar ratio (PLs: DSPE-PEG-2000)) DSPCPEGylated and DOPC-PEGylated liposomes using the thin film method, DSPC with DSPEPEG-2000 and DOPC with DSPE-PEG-2000 were first dissolved in 10 mL of ethanol. The organic solvent/ethanol was removed by rotary evaporation at 60 C and 50 C (above TT of

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respective PLs) in 30-40 min, respectively. When thin film was formed, it was allowed to dry in vacuum desiccator overnight. The following day, thin film was hydrated using 5 mL of normal saline at 60 C and 50 C to prepare DSPC-PEGylated and DOPC-PEGylated liposomes respectively. Sonication was then performed, using Grant XUB18 Ultrasonic Water Bath for 15 min, to see the effect on hydrodynamic particle size, PDI and morphology

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of liposomes before and after sonication. 2.7. Particle analysis 2.7.1. Mean size and distribution by dynamic light scattering (DLS) The hydrodynamic particles size and PDI of liposomes were measured by dynamic light scattering (DLS) Zeta-Sizer Nano ZS (Malvern, UK). Disposable cuvettes with 1 mL of the 5

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mM suspended liposomes were taken to measure particle size and PDI. All runs were performed in triplicate and each run comprised of 100 measurements at 25 °C. 2.7.2. Morphological analysis by cryo-TEM Liposome samples were imaged by cryo-TEM. The samples of prepared liposomes were applied to 300 mesh EM carbon coated film grids (EMS, Hatfield PA,USA) using a FEI

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Vitrobot Mark IV plunge freezer set (100% humidity) at 22 C. The samples were vitrified using thin ice and plunged by liquid ethane at -180 C. Frozen grids were cryo-transferred into a FEI Tecnai F30 transmission electron microscope (FEI, Einhoven, Netherlands) using a Gatan cryo holder. The microscope was operated at 300 kV and the samples were imaged at 179 C under low dose conditions using a Gatan K2 summit camera (Gatan, Pleasonton CA,

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USA). This was operated in counting mode at a dose rate of 9 e/px/s, a 10 s total exposure 10 s and a dose fractionation of 0.2 s. For acquisition of the image, Serial EM was used. The 50 frames were later motion corrected using the ‘Align’ function of Serial EM (Hyatt, 1984; Mastronarde, 2005; Vuitton, 2009). 2.8. Stability study

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Liposomes were tested for physical stability at different storage conditions. For this, that manufactured liposomes using both SCF and conventional methods i.e. DSPC-PEGylated and DOPC-PEGylated (5 mM with 94: 6 molar ratios in normal saline) were stored at -20 °C, 4 °C and 25 °C. To compare the results PDI and the hydrodynamic mean particle size (Zaverage) were recorded immediately after manufacturing and over a period of 3 months.

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2.9. Statistical analysis GraphPad Prism version 7.04 (GraphPad software Inc., San Diego, USA) was used and the data is presented as mean ± standard deviation (S.D). Two-way analysis of variance (ANOVA) was carried out, followed by Sidek post hoc multiple comparison test to determine the statistical difference among different groups. 13

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3. Results and discussion 3.1. Dispersibility of PLs in subcritical and supercritical CO2 The results showed that DSPC has the highest dispersibility in scCO2, while DPPG and DOPG showed “moderate” and “the least” dispersibility respectively (see Figure 2). The dispersibility of chemical material have a significant role in physiochemical interaction and processes. Different simulations, and calculative formulas have been developed previously, 250 to predict the solubility or dispersibility of different materials/compounds in scCO2 (Bian et al., 2015; Del Valle and Aguilera, 1988). Although prediction of solubility is helpful to some extent, its accuracy and application is not that much reliable though. Therefore, in the current study to determine the dispersibility of six types of PLs, a method was developed and for this birdseye view images of the SS vessel were collected before and after processing with 255 scCO2 at the assessed supercritical conditions. PLs, like DOPE, DOPC and DOTAP were not dispersed in scCO2 (were named as scCO2 non-dispersible PLs) thus a co-solvent i.e. ethanol used to manufacture liposomes (see following sections). In addition, it was also found that none of the PLs showed dispersibility at subcritical conditions in liquid CO, thus for the later experiments just supercritical conditions were used to manufacture liposomes using scCO 2602. The visual images taken at 0 min and after depressurization (at 60 min) of scCO2 SS vessel showed no change, while PLs such as DSPC, DOPG and DPPG showed different levels of dispersibility in scCO2 (Figure 2). It is fact that scCO2 has high solubility power because it has viscosity like that of liquid and diffusivity power like that of gases. The extent

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of dispersibility of dispersible PL’s (DSPC, DPPG and DOPG) was determined based on the visual changes and area of the SS vessel covered by layer of dispersed PLs. It is obvious from the Figure 2 that DSPC showed the highest dispersibility compared to DPPG (showed poor dispersibility) and DOPG (showed very poor dispersibility). The estimated semi-quantitative results of the visual dispersibility of the PLs in scCO2 are explained in the Table 2. DOPC

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was further selected to manufacture PEGylated liposomes, based on the better particle size range and PDI compared to conventional DOPE, and DOTAP liposomes and results are detailed in the following sections. It can be derived from the results that the higher the TT of PLs, the more they show dispersibility in scCO2. The more dispersibility shown by DSPC could be referred to strong

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bonds between long hydrocarbon chains and CO2 interactions. Such interactions are due to strong van der Waals forces and quadruple induced dipole with hydrocarbon chains of PLs. As a result, long fatty acyl chain helps CO2 to be surrounded by more hydrocarbon chains with improved distribution of PL molecules (Shin-ichiro et al., 2005; Zhao et al., 2015). In addition, cationic PL did not show any dispersibility, while zwitter-ionic and PLs with higher

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TT value i.e. DSPC showed a visible dispersibility.

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Figure 2. Birdseye view of SS vessel containing PLs, pre- and post-exposure to scCO2. Pre-exposures are (1a) DSPC, (2a) DPPG and (3a) DOPG and post-exposures to scCO2 are (1b) DSPC, (2b) DPPG and (3b) DOPG.

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Table 2. Visual estimation of dispersibility using a semi-quantitative/visual dispersibility of phospholipids (PLs) in scCO2. No. PLs Visual dispersibility in scCO2 1

DSPC

completely dispersible

2

DPPG

≥ 50 % dispersible

3

DOPG

≤ 50% dispersible

4

DOPC

not dispersible

5

DOPE

not dispersible

6

DOTAP

not dispersible

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3.2. Manufacturing conventional (non-stealth) liposomes using SCF technology In the first set of experiments, at the highest density (893 kg/dm3 based on pure CO2) at 37 °C and 250 bar, the conventional/non-PEGylated liposomes were manufactured using six types of PLs and the results of particle size are shown in Figure 3 A and B. The effect of different 295

conditions on particle size of manufactured liposomes using scCO2 explained in the following sections. 3.3. Asses the effect of transition temperatures The effect of varying temperature showed that below TT (See Table 1 for TT values and Figure 3 A for results) with 250 bar pressure liposomes had showed nano-range, for both

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DSPC and DPPG PLs and particle size was significantly lower (i.e. **, where **: P ≤ 0.0) compared to the liposomes manufactured above TT of the used PLs (see Table 1 for TT values). The lesser of density on increasing the temperature is one of the reason, which result in reduction of the solubility power of scCO2 and hence end in increase of the particle size of the liposomes. In addition, the SCF generally does not follow the ideal gas law, meaning that

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the trend of increase/decrease in viscosity and density behaves differently from normal fluids and varies at every point upon changing the temperature and pressure (Span and Wagner, 1996).

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3.4. Effect of co-solvent The ethanol, as a co-solvent with scCO2, used to assess its effect on (PLs e.g. DOPE, DOPC, 310

DOTAP) solubility (of scCO2) and particle size of liposomes. The results of the particle size analysis are presented in Figure 3 B. Based on the initial experiments of dispersibility, (as described in the above sections) three PLs i.e. DOPE, DOPC and DOTAP did not show dispersibility in scCO2, while DOPG showed “least” dispersibility. DOPE (inhibit endosomal reuptake), and DOPC best suited for high drug loading and delivery of hydrophobic drugs,

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while DOTAP (cationic), improve targeting of negatively charged cancer cells with enhanced gene and DNA delivery effect (Zhang et al., 2004). At the end of depressurization suspended/dissolved particles precipitated out after depressurisation (and proliposomes were formed) which were hydrated to manufacture liposomes. Atomization and nucleation effect during scCO2 processing with the aid of ethanol provided a uniform sized particle distribution

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with nanoparticle size range (See Figure 3 B). The hydrodynamic particle size of DOPC, DOPE and DOTAP liposomes was 557±17 nm, 642.5±44.5 nm and 233±25.5 nm respectively. DOPC was further selected (from non-dispersible PLs) to prepare stealth/PEGylated liposomes and to see the effect on particle size and morphology. 3.5. Effect of sonication

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The results of the effect of sonication on particle size of liposomes are shown in Figure 3. It was observed for DOPE, DOPC, DOPG and DOTAP conventional liposomes, the particle size was significantly reduced (i.e. ***, where ***: P ≤ 0.001) after 15 min sonication, which proved that sonication at the assessed conditions helped to reduce the particle size of liposomes. The nano-sized liposomes are formed due to the formation of tiny

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droplets called proliposomes. This could be due to interaction among the PL mixture, scCO2 and ethanol.

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Figure 3. Particle size analysis of conventional liposomes using scCO2 at 250 bar, 200 RPM and 60 min (n=3 ±S.D) before and after sonication. (A) DSPC and DPPG liposomes using scCO2 below TT (37°C) and above TT (56 °C for DSPC and 42 °C for DPPG). (B) DOPG, DOPE, DOPC and DOTAP liposomes using scCO2 at 37°C. Where **: P ≤ 0.01 and *** P ≤ 0.001 (Two way ANOVA with Sidek post hoc test).

3.6. Manufacturing PEGylated (stealth) liposomes using SCF technology 340

The particle size, morphological analysis and the stability studies of the PEGylated liposomes manufactured using SCF technology are described in the following sections. The process yield was also calculated using SCF method and it was found to be 83.65 %. This could be associated due to depressurization at high pressure when SCF is released at last step then it may take some particles outside of the vessel. This yield could be improved on large

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scale SCF processing. Effects of different varying conditions on DSPC (a scCO2 dispersible PL) and DOPC (non-dispersible PL in scCO2) PEGylated liposomes is explained in the following sections. 3.6.1. Effect of varying temperature and pressure The results of liposomes manufactured using different conditions such as: pressure

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and temperature explained in Figures 4-5. It has been shown that at 37 °C and 250 bar the liposomes had nano-range with better PDI of nanoparticles/liposomes. This could be due to 19

high density of CO2 i.e. 893 kg/dm3 at these conditions, which is very close to density of its liquid and show high solubility. The higher density increases the solvation power of scCO2 and thus increases mass transfer rate, which reduces the particle size of liposomes. It has been 355

shown that the particle size of liposomes at 250 bar was significantly lower (see Figures 4-5, for significant P values) than the other pressures (150, 200 and 300 bar) and the temperature below TT reduced particle size range effectively, compared to the liposomes manufactured at temperature above TT. The reduction in density of scCO2 on increasing pressure has been described previously (Span and Wagner, 1996). So increasing the temperature did not

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increase the density (i.e. solubility would reduce on increasing temperature at fixed pressure of 250 bar) and hence particle size was bigger than the particle size of liposomes manufactured at lower temperature. 3.6.2. Effect of time and sonication The effect of time and sonication has been shown in Figures 4-5. It is obvious from

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Figures that more experimental time i.e. 60 min provided better results in terms of particle size and PDI compared to 30 min. In addition, the effect of sonication on particle size of liposomes played a vital role and significantly reduced the particle size and PDI of liposomes. The particle size of liposomes is inversely proportional to processing time and has been reported elsewhere in the literature (Span and Wagner, 1996), which states that lengthy

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processing time reduces the particle size more effectively. At constant temperature, the increase in the pressure did not help in the reduction of particle size of DSPC-PEG and DOPC-PEG liposomes.

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Figure 4. Particle size optimization of DSPC-PEGylated liposomes using scCO2 at varying conditions of temperature, time and pressure and effect of sonication (n=3 ± S.D). (A) DSPCPEG liposomes at 150, 200 and 250 bar after 30 min, below TT at 37 °C (for all pressures) and above TT at 56°C (for 250 bar). (B). DSPC-PEG liposomes at 150, 200, 250 and 300 bar after 60 min below TT at 37 °C (for all pressures) and above TT at 56°C (for 250 bar). Where *: P ≤ 0.05 and ** P ≤ 0.01 (Two way ANOVA with Sidek post hoc test).

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Figure 5. Particle size optimization of DOPC-PEGylated liposomes using scCO2 at varying conditions of temperature, time and pressure and effect of sonication (n=3 ± S.D). (A) DOPCPEG liposomes at 150, 200 and 250 bar after 30 min and 37 °C. (B). DOPC-PEG liposomes at 150, 200, 250 and 300 bar after 60 min and 37 °C. Where * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 (Two way ANOVA with Sidek post hoc test).

3.7. 390

Manufacturing liposomes using thin film method and comparison with SCF technology

The results of the PEGylated liposomes manufacture using thin film method are explained and compared with SCF technology in the following sections. 3.7.1. Particle size characterization The particle size and PDI of the DSPC-PEG and DOPC-PEG liposomes manufactured using 395

thin film and its comparison with SCF technology using scCO2 (with high density at 250 bar and 37 °C) is described in Table 3. The mean particle size diameter of DSPC-PEG and DOPC-PEG liposomes using thin film method was 274.4±16.5 nm and 131.3±3.4 nm respectively. The data shows that the mean particle size of the liposomes i.e. DSPC-PEG and DOPC-PEG liposomes using SCF technology at 250 bar and 37 °C was 98.3±3.3 nm and

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124.5±4.1 nm respectively. The particle size and PDI of the SCF processed liposomes was

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relatively smaller, compared to conventional thin film method. (Table 3). The better PDI results show that SCF assisted DSPC-PEG liposomes were homogenous with narrow range of particle sizes, while liposomes manufactured using thin film method were of different sizes/heterogeneous and size distribution graph also support and show the multiple size 405

populations of nanoparticles. The further reduction of particle size and PDI could be achieved in conventional methods by using: sonication, homogenization or extrusion techniques. Whereas particle size and PDI of the liposomes obtained using SCF method avoids the use of such techniques for reduction of particle size and PDI (Park et al., 2012; Wagner and Vorauer-Uhl, 2011). Moreover, SCF as a green technology could be better alternative to

410

traditional used methods, which would save time, avoid use large amounts of energy and organic solvents, and also save environment from waste products (as produced by conventional Bangham method). The particle sizing data and morphology of liposomes was further confirmed by electron microscopy, detailed in the following section.

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415

Table 3. Comparison of DSPC-PEGylated and DOPC-PEGylated liposomes manufactured using SCF and Bangham method.

No. 1 2 3

Thin Film

Composition and conditions (PL + polymer + solvent/co-solvent & volume) DSPC and DSPE-PEG-2000 (94:6) + 10 mL ethanol

SCF

Method

Total time for Mean Particle preparation of Size ± S.D liposomes (h) (n=3)

Mean PDI ± S.D (n=3)

24 – 32

153.6 ± 4.5

0.4 ± 0.02

DSPC and DSPE-PEG-2000 (94:6) 250 bar, 37 °C, 200 RPM

2

98.3 ± 3.3

0.2 ± 0.01

Thin Film

DOPC and DSPE-PEG-2000 (94:6) + 10 mL ethanol

24 - 32

131.3 ± 3.4

0.3 ± 0.01

SCF

DOPC and DSPE-PEG-2000 (94:6) + 0.5 mL ethanol, 250 bar, 37 °C, 200 RPM

2

124.5 ± 4.1

0.2 ± 0.01

4

24

3.7.2. Morphological analysis using cryo-TEM The images taken by cryo-TEM are shown in Figures 6- 7. Results showed that DSPC and DOPC stealth/PEGylated liposomes manufactured using thin film and SCF technology were 420

spherical and nano-vesicles with absence of any aggregation. The particle size and homogeneity observed by Cryo-TEM was similar to the results obtained by Zeta-sizer, with a slight difference. This difference in the particle size obtained using DLS and Cryo-TEM might be due to hydration shells surrounding liposomes, and show a bit larger particle size using DLS machine when compared to the results obtained from cryo-TEM. The effect on

425

differences in the particle size due to hydration shells is well explained in the literature (Mahl et al., 2011; Müller et al., 2004). The results showed that liposomes have unilamellar structure for both SCF and thin film method but that manufactured using SCF method are more smaller and have lower PDI compared to that manufactured using the thin film method. This nanosize range could better help to cross the cell membranes and targeting the cancerous

430

cells, which would help to achieve increased intracellular uptake. The size distribution graph in Figure 6 C, 6 D of DSPC-PEGylated liposomes showed that liposomes manufactured using SCF method were homogenous with single population (PDI: 0.2± 0.01), whereas liposomes manufactured using thin film method are heterogeneous with multiple sized populations (PDI: 0.4± 0.02). The improved particle size reduction of SCF assisted liposomes is

435

associated with process of atomization and nucleation too. This could be reason when thin film is hydrated then liposomes manufactured using SCF process are more better, and homogenously dispersed as explained in the detail in the above sections. SCF of CO2 is reason of atomization and nucleation of the liposomal nanoparticles and after hydration; the manufactured liposomes were of better PDI with more reduction in the particle size compared

440

to thin film liposomes. SCF technology could provide a better alternative approach to

25

manufacture liposomes with number of benefits such as, use of less amount of organic solvents, cheap, rapid and less intensive technique.

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Figure 6. Cryo-TEM images and size distribution data by DLS of DSPC-PEGylated liposomes. (A) Cryo- TEM image of DSPC-PEG liposomes prepared using thin film method (B) Cryo- TEM image of DSPC-PEG liposomes, prepared using SCF technology at 250 bar, 37 °C, 200 RPM processed for 1 h. (C) Particle size distribution graph of thin film-DSPC-PEG liposomes by Zetasizer (DLS machine). (D) Particle size distribution graph of SCF-DSPC-PEG liposomes by Zetasizer (DLS machine).

450

26

455

Figure 7. Cryo-TEM images and size distribution data by DLS of DOPC-PEGylated liposomes (A) Cryo- TEM image of DOPC-PEG liposomes prepared using thin film method (B) Cryo- TEM image of DOPC-PEG liposomes, prepared using SCF technology at 250 bar, 37 °C, 200 RPM processed for 1 h. (C) Particle size distribution graph of Thin film-DOPC-PEG liposomes by Zetasizer (DLS machine). (D) Particle size distribution graph of SCF-DOPC-PEG liposomes by Zetasizer (DLS machine). 3.8. Stability study

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The difference between stability results of the particle size and PDI of the liposome stored at 4 °C or – 20 °C was very less after 3 months. However, room temperature did not show promising results in terms of stability of the liposomes. The results showed that liposomes are best stable at 4 °C or – 20 °C (Table 4). While comparing the stability of DSPC-PEGylated liposomes manufactured using SCF method with that of thin film method, it can be seen that

465

liposomes manufactured using SCF technology showed better stability. Summarizing this, the changes observed in the particle size were minimal by SCF liposomes and were more stable compared to thin film method. These stability results also match with that obtained by Otake

27

et al (Otake et al., 2006) and some other researcher (Aburai et al., 2011; Kadimi et al., 2007). Static repulsion of the carbonic acids incorporated into the bilayer membrane, well explain 470

the long-term stability of liposomes prepared using scCO2 (Bothun et al., 2005). Liposomes manufactured using thin film showed more increase in the particle size, and one of the possible reasons could be oxidation of polyunsaturated acyl chains of lipids and hydrolysis of ester bonds.

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Table 4. Stability of DSPC-PEGylated and DOPC-PEGylated liposomes over the period of 3 months stored at different temperatures. Liposomes

Particle Size (nm)

Storage (°C)

Initial

Particle Size (nm) After 1 Month

After 2 Months

After 3 Months

DSPCPEG- Film

153.6 ± 4.5

4±2 -20 ± 2 25 ± 2

157.8 ± 3.5 159.1 ± 4.34 161.7 ± 3.4

156.5 ± 5.4 163.8 ± 3.2 165.1 ± 4.6

158.2 ± 3.5 167 ± 5.25 166.6 ± 4.6

DSPCPEG- SCF

98.3±3.3

4±2 -20 ± 2 25 ± 2

99.8 ± 2.5 100.8 ± 4.3 102.8 ± 3.4

98.3.5 ± 4.74 102.3 ± 2.6 103.4 ± 5.1

100.7 ± 3.8 102.4 ± 4.6 104.3 ± 2.8

DOPCPEG- Film

131.3±3.4

4±2 -20 ± 2 25 ± 2

132.5 ± 5.5 136.2 ± 2.6 138.4 ± 4.9

137.5 ± 5.6 136.6 ± 3.31 136.5 ± 4.9

140.4 ± 4.6 138.8 ± 3.8 145.4 ± 4.3

DOPCPEG- SCF

124.5±4.1

4±2 -20 ± 2 25 ± 2

122.4 ± 2.5 127.2 ± 3.5 126.6 ± 3.8

125.5 ± 3.6 126.6 ± 5.4 127.2 ± 2.8

126.4 ± 3.6 128.8 ± 2.9 131.4 ± 3.9

Mean= ±S. D, n=3

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480

4. Conclusion In our study, the method was developed to determine the dispersibility of PLs and to manufacture conventional liposomes from six types of PLs using SCF technology. The two PLs (DPSC and DOPC) were selected to manufacture PEGylated/stealth liposomes using scCO2 and results were compared with the liposomes manufactured using the thin film

485

method. It was observed that DSPE-PEG2000 caused reduction of particle size and PDI. The method was optimized for best combination of processing time, temperature and pressure, to manufacture liposomes and it was found that 37 °C, 250 bar for 1 h provided best results in terms of particle size and morphology. DLS data and cryo-TEM images showed that SCF mediated liposomes were better in particle size and morphology when compared with the thin

490

film method. The stability of particle size at various temperatures was tested which shows that DSPC-PEG liposomes manufactured using SCF technology were more stable over the period of 3 months. The data from this study provides original information about the dispersibility and manufacturing of liposomes using SCF green technology and a better alternative to traditional used methods; it is less intensive, rapid, nontoxic and easily scalable.

495

Our study provides a good starting point for future formulation studies and to manufacture drug loaded liposomes using SCF technology. 5. Author information All authors approved the final version of the manuscript. Author Contributions

500

James Falconer and Peter Moyle conceived and designed the project. Faheem Maqbool performed the experiments and wrote the manuscript. Kristofer Thurecht provided conceptual input and revised manuscript.

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Acknowledgement 505

Faheem Maqbool is a recipient of Australian Government Research Training Program Scholarship from The University of Queensland, Brisbane, Australia. The authors thank Professor Andrew K. Whittaker of the Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072 Australia, for his support and access to specialized equipment, including the SCF unit used in this research.

510

Conflict of interest The authors declare no conflict of interest.

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