Accepted Manuscript

Dispersibility of phospholipids and its optimization for efficient production ofliposomes using supercritical fluid technology

Faheem Maqbool, Peter M. Moyle, Kristofer J. Thurecht, James R. Falconer

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

To appear in: International Journal of Pharmaceutics

Received Date: 2 February 2019Revised Date: 23 March 2019Accepted Date: 25 March 2019

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

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1

Dispersibility of phospholipids and its optimization for efficient production

of liposomes using supercritical fluid technology

Faheem Maqbool1, Peter M. Moyle

1, *, Kristofer J. Thurecht

2, James R. Falconer

1, *

1School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia

2The Centre for Advanced Imaging (CAI), The University of Queensland, Brisbane, QLD

4072, Australia

*Co-correspondence:

j.falconer@uq.edu.au (J.R.F) Tel: +61(4) 312 73844

and

p.moyle@uq.edu.au (P.M.M.) Tel: +61(7) 334 61869

2

Graphical Abstract:

3

Abstract

Liposomes are promising delivery vehicles and offer the added drawcard of being able to be 5

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-

phosphocholine (DSPC) showed highest post-processing dispersibility, while 1,2-dioleoyl-sn-10

glycero-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

narrowest polydispersity index (PDI) and spherical shaped as shown by cryo-transmission 15

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

-20 °C) using SCF technology was better over a period of 3 months. The current study would 20

provide green alternative method, less laborious, save time and energy.

Keywords: Green technology, Zwitter-ionic, Electron microscopy, Particle engineering, Drug

delivery, Size distribution

4

1. Introduction 25

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

membranes in the human body. Liposomes can be unilamellar or multilamellar, based on the 30

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.

Liposomes have had commercial success in delivering anticancer drugs at cancerous 35

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

et al., 2013). Liposomes take advantage of tumor leaky microenvironment; thus liposomal 40

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

system. The effects of polymers associated with different PLs (e.g. charge, liposome particle 45

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 non-

toxic and biodegradable within the human body (Gregoriadis and Ryman, 1971; Liu and

Boyd, 2013; Sharma and Sharma, 1997).

5

Several methods have been developed to produce liposomes. These include the 50

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-

formation/homogenisation, and emulsion methods (Batzri and Korn, 1973; Deamer and 55

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

use of organic solvent, thus a green/er technology, and operating can use cheap, readily 60

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

likely to cause degradation to a thermolabile compound and only requires a small amount of 65

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

technologies can be scaled up, so simpler lab-bench equipment is sufficient for optimising 70

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.

6

Although, use of acCO2 has been previously reported in many studies for natural product 75

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

scCO2 and production of liposomes using these PLs and SCF technology. Although some 80

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.

The aim of this study was to determine the dispersibility of six different charged PLs 85

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.

1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), e. DOTAP, f. DOPE. (See 90

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;

varying temperature, pressure, time, and their effects were evaluated on the particle size, PDI 95

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.

7

2. Materials and methods 100

2.1. Materials

DSPC, 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2-dioleoyl-sn-

glycero-3-phospho-(1'-rac-glycerol) (DOPG), DOPE, DOPC, DOTAP and 1,2-distearoyl-sn-

glycero-3-phospho- ethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)

(DSPE-PEG-2000) were purchased from Avanti Polar Lipids, Alabama, USA. Ethanol and 105

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.

110

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

capacity) was used as reaction/processing vessel for the experiment. At first, the SS vessel 115

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

vessel used to rotate the peddle (hanging in the high-pressure SS vessel) for mixing and 120

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

8

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

CO2 gas from scCO2, which was then released back into the air. 125

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

scCO2 in separate experiments. For all experiments 20 ± 0.1 mg of the PLs were used in 130

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 high-

pressure 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

assess/determine the dispersibility of PLs in scCO2 bird-eye images of the inner section/lining 135

of SS vessel were collected before processing (0 min) and after depressurization.

9

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. 140

2.4. Manufacturing conventional liposomes using SCF technology

The SCF technology was first used in the current study to manufacture conventional/non-

stealth liposomes of the all PLs (See Table 1). To manufacture 5 mM (94: 6 molar ratios

(PLs: DSPE-PEG-2000) of conventional liposomes of six PLs, the experiments were 145

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

10

proliposomes, 5 mL of the normal saline was added into the high pressure stainless steel 150

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

To see the effect of temperatures above TT of the PLs, additional experiments were 155

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

To assess the effect of co-solvents with scCO2 on the dispersibility of PLs, such as: DOPC, 160

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

depressurized of the of SS vessel proliposomes were hydrated using 5 mL of normal saline 165

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

Bath for 15 min. To analyse these effects the particle size analysis was performed of the 170

manufactured liposomes before and after sonication and compared accordingly.

11

Table 1. Transition temperatures and surface charges of the selected phospholipids (PLs)

used in the study.

175

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

scCO2, provided further direction to manufacture PEGylated liposomes. The composition of 180

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

DOPC PL, 0.5 mL of ethanol was used as co-solvent with 60 mL of scCO2. The unsaturated 185

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

modifications was studied and is explained in the following sections. 190

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

at constant temperature i.e. 37 °C in separate experiments. Moreover, DSPC-PEGylated 195

Type of PLs DSPC DOPE DOPC DOTAP DPPG DOPG

Charge on PLs

Zwitter-

ionic

Zwitter-

ionic

Zwitter-

ionic

Cationic Anionic Anionic

Transition

Temperature (°C)

55 -16 -17 5 41 -18

12

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,

separate experiments were performed at 30 and 60 min with similar pressure and temperature 200

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.

2.6. Manufacturing PEGylated liposomes using thin film (Bangham) method 205

To prepare (5 mM of liposomes with 94: 6 molar ratio (PLs: DSPE-PEG-2000)) DSPC-

PEGylated and DOPC-PEGylated liposomes using the thin film method, DSPC with DSPE-

PEG-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

respective PLs) in 30-40 min, respectively. When thin film was formed, it was allowed to dry 210

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

of liposomes before and after sonication. 215

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

13

mM suspended liposomes were taken to measure particle size and PDI. All runs were 220

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

Vitrobot Mark IV plunge freezer set (100% humidity) at 22 C. The samples were vitrified 225

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,

USA). This was operated in counting mode at a dose rate of 9 e/px/s, a 10 s total exposure 10 230

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

Liposomes were tested for physical stability at different storage conditions. For this, that 235

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 (Z-

average) were recorded immediately after manufacturing and over a period of 3 months.

2.9. Statistical analysis 240

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.

14

3. Results and discussion 245

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 scCO2 255

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 scCO2. 260

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

of dispersibility of dispersible PL’s (DSPC, DPPG and DOPG) was determined based on the 265

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

15

was further selected to manufacture PEGylated liposomes, based on the better particle size 270

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

bonds between long hydrocarbon chains and CO2 interactions. Such interactions are due to 275

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

TT value i.e. DSPC showed a visible dispersibility. 280

16

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. 285

17

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

290

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

conditions on particle size of manufactured liposomes using scCO2 explained in the following 295

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

DSPC and DPPG PLs and particle size was significantly lower (i.e. **, where **: P ≤ 0.0) 300

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

the trend of increase/decrease in viscosity and density behaves differently from normal fluids 305

and varies at every point upon changing the temperature and pressure (Span and Wagner,

1996).

18

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,

DOTAP) solubility (of scCO2) and particle size of liposomes. The results of the particle size 310

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,

while DOTAP (cationic), improve targeting of negatively charged cancer cells with enhanced 315

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

with nanoparticle size range (See Figure 3 B). The hydrodynamic particle size of DOPC, 320

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

The results of the effect of sonication on particle size of liposomes are shown in 325

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

droplets called proliposomes. This could be due to interaction among the PL mixture, scCO2 330

and ethanol.

19

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, 335

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

The particle size, morphological analysis and the stability studies of the PEGylated 340

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

scale SCF processing. Effects of different varying conditions on DSPC (a scCO2 dispersible 345

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

and temperature explained in Figures 4-5. It has been shown that at 37 °C and 250 bar the 350

liposomes had nano-range with better PDI of nanoparticles/liposomes. This could be due to

20

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

shown that the particle size of liposomes at 250 bar was significantly lower (see Figures 4-5, 355

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

increase the density (i.e. solubility would reduce on increasing temperature at fixed pressure 360

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

Figures that more experimental time i.e. 60 min provided better results in terms of particle 365

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

processing time reduces the particle size more effectively. At constant temperature, the 370

increase in the pressure did not help in the reduction of particle size of DSPC-PEG and

DOPC-PEG liposomes.

21

375 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) DSPC-

PEG 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 380

*: P ≤ 0.05 and ** P ≤ 0.01 (Two way ANOVA with Sidek post hoc test).

22

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) DOPC-

PEG liposomes at 150, 200 and 250 bar after 30 min and 37 °C. (B). DOPC-PEG liposomes at 385

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. Manufacturing liposomes using thin film method and comparison with SCF

technology 390

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

thin film and its comparison with SCF technology using scCO2 (with high density at 250 bar 395

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

124.5±4.1 nm respectively. The particle size and PDI of the SCF processed liposomes was 400

23

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

populations of nanoparticles. The further reduction of particle size and PDI could be achieved 405

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

traditional used methods, which would save time, avoid use large amounts of energy and 410

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.

24

Table 3. Comparison of DSPC-PEGylated and DOPC-PEGylated liposomes manufactured using SCF and Bangham method. 415

No. Method

Composition and conditions

(PL + polymer + solvent/co-solvent &

volume)

Total time for

preparation of

liposomes (h)

Mean Particle

Size ± S.D

(n=3)

Mean PDI ± S.D

(n=3)

1 Thin

Film

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

ethanol 24 – 32 153.6 ± 4.5 0.4 ± 0.02

2 SCF

DSPC and DSPE-PEG-2000 (94:6) 250 bar,

37 °C, 200 RPM 2 98.3 ± 3.3 0.2 ± 0.01

3 Thin

Film

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

ethanol 24 - 32 131.3 ± 3.4 0.3 ± 0.01

4 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

25

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

spherical and nano-vesicles with absence of any aggregation. The particle size and 420

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

differences in the particle size due to hydration shells is well explained in the literature (Mahl 425

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

cells, which would help to achieve increased intracellular uptake. The size distribution graph 430

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

associated with process of atomization and nucleation too. This could be reason when thin 435

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

to thin film liposomes. SCF technology could provide a better alternative approach to 440

26

manufacture liposomes with number of benefits such as, use of less amount of organic

solvents, cheap, rapid and less intensive technique.

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 445

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

27

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 455

Zetasizer (DLS machine). (D) Particle size distribution graph of SCF-DOPC-PEG liposomes by

Zetasizer (DLS machine).

3.8. Stability study

The difference between stability results of the particle size and PDI of the liposome stored at 460

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

liposomes manufactured using SCF technology showed better stability. Summarizing this, the 465

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

28

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

the long-term stability of liposomes prepared using scCO2 (Bothun et al., 2005). Liposomes 470

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.

475

Table 4. Stability of DSPC-PEGylated and DOPC-PEGylated liposomes over the period of 3

months stored at different temperatures.

Mean= ±S. D, n=3

Liposomes Particle Size (nm) Storage

(°C)

Particle Size (nm)

Initial After 1 Month After 2

Months

After 3

Months

DSPC-

PEG- Film

153.6 ± 4.5 4 ± 2 157.8 ± 3.5 156.5 ± 5.4 158.2 ± 3.5

-20 ± 2 159.1 ± 4.34 163.8 ± 3.2 167 ± 5.25

25 ± 2 161.7 ± 3.4 165.1 ± 4.6 166.6 ± 4.6

DSPC-

PEG- SCF

98.3±3.3 4 ± 2 99.8 ± 2.5 98.3.5 ± 4.74 100.7 ± 3.8

-20 ± 2 100.8 ± 4.3 102.3 ± 2.6 102.4 ± 4.6

25 ± 2 102.8 ± 3.4 103.4 ± 5.1 104.3 ± 2.8

DOPC-

PEG- Film

131.3±3.4 4 ± 2 132.5 ± 5.5 137.5 ± 5.6 140.4 ± 4.6

-20 ± 2 136.2 ± 2.6 136.6 ± 3.31 138.8 ± 3.8

25 ± 2 138.4 ± 4.9 136.5 ± 4.9 145.4 ± 4.3

DOPC-

PEG- SCF

124.5±4.1 4 ± 2 122.4 ± 2.5 125.5 ± 3.6 126.4 ± 3.6

-20 ± 2 127.2 ± 3.5 126.6 ± 5.4 128.8 ± 2.9

25 ± 2 126.6 ± 3.8 127.2 ± 2.8 131.4 ± 3.9

29

4. Conclusion 480

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

method. It was observed that DSPE-PEG2000 caused reduction of particle size and PDI. The 485

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

film method. The stability of particle size at various temperatures was tested which shows 490

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.

Our study provides a good starting point for future formulation studies and to manufacture 495

drug loaded liposomes using SCF technology.

5. Author information

All authors approved the final version of the manuscript.

Author Contributions

James Falconer and Peter Moyle conceived and designed the project. Faheem Maqbool 500

performed the experiments and wrote the manuscript. Kristofer Thurecht provided conceptual

input and revised manuscript.

30

Acknowledgement

Faheem Maqbool is a recipient of Australian Government Research Training Program 505

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.

Conflict of interest 510

The authors declare no conflict of interest.

31

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