J.

of Supercritical Fluids 165 (2020) 104984

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

journal homepage: www.elsevier.com/locate/supflu

Sustainable technologies for liposome preparation

Maja Leitgeb a,b,∗ , Željko Knez a,b , Mateja Primožič a
a
University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova Ulica 17, Maribor, 2000, Slovenia
b
University of Maribor, Faculty of Medicine, Taborska Ulica 8, Maribor, 2000, Slovenia

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• Sustainable green technologies for

liposome preparation.
• Production of micro- and nano-sized
liposomes with narrow size distribu-
tion.
• Supercritical fluids assisted methods
improve liposome encapsulation effi-
ciency.

a r t i c l e i n f o a b s t r a c t

Article history: Liposome possesses a great number of advantages and therefore they can be used for a variety of appli-
Received 14 April 2020 cations. Among other things, they can serve as useful drug carriers in preclinical and clinical trials.
Received in revised form 19 June 2020 Supercritical fluids assisted technology is an appropriate method for liposome preparation because of its
Accepted 5 July 2020
nontoxicity to the environment enables particle size manipulation and solvent-free production. Thus, the
Available online 11 July 2020
use of supercritical fluids (SCFs) provides advanced technology for liposome preparation and may become
the dominant technology for their preparation in the future. This review discusses the classification of
Keywords:
liposome and gives a short overview of their applications and conventional methods of preparation.
Liposome
Preparation
Emphasis is placed on the use of various supercritical fluids assisted methods for liposome prepara-
Supercritical fluids tion and their advantages and disadvantages. The reader is also updated about recent developments in
Supercritical fluids assisted methods supercritical fluids assisted liposome production technology.
© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Classification of liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Liposome as a drug delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.1. Commercially available formulations of liposome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. Methods of drug loading in liposome preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5. Conventional techniques for liposome preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5.1. Tools for size reduction and uniform size distribution of liposome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

∗ Corresponding author at: University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova ulica 17, Maribor, 2000, Slovenia.
E-mail addresses: maja.leitgeb@um.si (L. Maja), zeljko.knez@um.si (K. Željko), mateja.primozic@um.si (P. Mateja).

https://doi.org/10.1016/j.supflu.2020.104984
0896-8446/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
2 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984

6. Supercritical fluids (SCFs) methods for liposome preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

6.1. Depressurization of an expanded liquid organic solution-suspension (DELOS) method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6.2. Supercritical anti-solvent (SAS) method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6.3. Rapid expansion of supercritical solutions (RESS) method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6.4. Supercritical assisted liposome formation (SuperLip) method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6.5. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6.6. Batch or continuous SCF processes for liposome preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1. Introduction determined using different analytical methods (UV–vis detection,

HPLC, GC, GC–MS etc.) depending on the active compound which
Liposomes are colloidal, vesicular structures, composed of is incorporated in liposomes. When the incorporated substance is
one or more lipid bilayers surrounding an equal numbers protein, direct and indirect methods for EE determination could be
of aqueous compartments [1]. Liposome can be made from used. While in direct methods, the EE of encapsulation is calculated
natural substances and are therefore nontoxic, biocompati- directly from the loaded protein in the nanoparticles, in the indirect
ble, biodegradable and non-immunogenic. Phosphatidylcholine method, the non-entrapped protein or drug is considered [16]. The
(PC), phosphatidylglycerol (PG), dipalmitoylphosphatidylcholine liposome preparation methods can be categorized as conventional
(DPPC), distearoylphosphatidylcholine (DSPC), cholesterol (CH), and novel methods (e.g. supercritical assisted method for liposome
stearylamine, dicetylphosphat, or a mixture of these are usually preparation). There are already some review articles where SCFs
the composites of the bilayer [2]. technologies for liposome preparation are described [17–20], but
Liposome properties are mostly dependent on lipid composi- in this review an emphasis on the impact of individual SCFs tech-
tion, surface charge, size, and the method of preparation [3]. The nology on the size, types of vesicles and EE of prepared liposomes
rigidity and the charge of the bilayer are conditioned by the choice including lipid/active ingredients of liposome preparation prepared
of the bilayer components. Unsaturated PC species from natural by defined SCFs method is done. It briefly explains the classifica-
sources (egg or soybean PC) give highly permeable and less stable tion of liposome and their potential applications, with a focus on
bilayers, while more rigid and impermeable bilayer structures are novel techniques for liposome formation using supercritical fluid
obtained when lipid saturated phospholipids (PLs) with long acyl assisted technologies.
chains (such as DPPC) are used [3–6]. Liposome possesses a great
number of features and therefore they can be used for a variety of 2. Classification of liposomes
applications. The general application of liposome may be attributed
to their use in respiratory and eye disorders, vaccine adjuvants, Liposome formation enables the amphiphilic character of lipids
brain targeting and anti-infective agents, and their clinical appli- which, with the force of hydrophobic interaction, assemble into
cations include cancer treatment, antimicrobial therapy and gene bilayers. Three groups of liposome exist by their morphology, using
therapy [7–9]. conventional classification: multilamellar vesicle (MLV), small
Liposome can serve as carriers for drugs or other macro- unilamellar vesicle (SUV), large unilamellar vesicle (LUV) and mul-
molecules for delivery into human and animal bodies, since they tivesicular vesicle (MVV) [21] (Fig. 2). These different vesicular
simplify site-specific drug delivery, e.g. to tumor tissues [10] and structures can be achieved from the self-assembly of a certain
are therefore of great interest to the pharmaceutical and cosmetic amphiphilic molecule. Because of their size, amphiphilic character,
industries. Liposomes are also attractive for applications in the food and biocompatibility, liposomes have been widely used as biomem-
and farming industries, since they can be used for encapsulation branes as well as delivery vehicles for various bioactive materials.
of unstable compounds (for example, antimicrobials, antioxidants, However, for drug encapsulation, size and homogeneity of the
flavors and bioactive substances) resulting in better stability of liposomes are more important than the number of lamellars in a
them. Liposome can trap both hydrophobic and hydrophilic com- liposome [14]. Therefore, for vesicles used as drug delivery systems,
pounds, prevent their decomposition, and release the entrapped the membrane composition and supramolecular organization are
compounds at selected targets [11–13]. Therefore, they offer a pow- highly important. For drug delivery applications, the desirable size
erful solubilizing system for a wide range of compounds. of liposome ranges between 50 and 200 nm [22,23].
Liposome exhibit many special biological characteristics,
including (specific) interactions with biological membranes and 3. Liposome as a drug delivery system
various cells, as a result of their physico-chemical properties [14].
Advantages and disadvantages of conventional liposome applica- Drug carriers can provide benefits over traditional drug formu-
tions are depicted in in Fig. 1. Industrial applications of liposomes lations [25]. They protect the entrapped drug from degradation and
are generally focused on drug delivery system and as adjuvants premature metabolization during release in the human body [26].
in vaccination in medicine as well as support matrix for various Liposomes belong to the vesicular delivery systems that can deliver
ingredients and penetration enhancer in cosmetics. Furthermore, hydrophilic or hydrophobic substances. They are spherical, have
liposomes are used as signal enhancers/carriers in medical diag- small particle size and high drug EE, possess an inner aqueous core
nostics and analytical biochemistry, as solubilizers for various and an external double lipidic layer [27] and are biocompatible with
ingredients etc. [14]. human cells [28–30].
Liposomes can be prepared using a wide range of methods which The first liposomes were introduced in 1965 by Alec Bang-
influence the liposome characteristics, such as size, lamellarity ham [31]. As excellent carrier systems because of their similarity
and encapsulation efficiency (EE, which is expressed as the per- to natural cells [19] they are able to overcome biological barri-
centage of drug that is successfully entrapped into the liposome). ers [32]. Research on liposome technology has progressed from
The concentration of incorporated substances in liposomes can be conventional vesicles to “second-generation liposome”, in which
 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984 3

Fig. 1. Advantages and disadvantages of conventional liposome applications [3,15].

Fig. 2. Types of vesicles depending on size and lamellarity. SUV, small unilamellar vesicle (20–100 nm); LUV, large unilamellar vesicle (>100 nm); MLV, multilamellar vesicle
(>500 nm); MVV multivesicular vesicle (>1000 nm) [21,24].

long-circulating liposomes are obtained by modulating the lipid Table 1

Advantages and disadvantages of stealth liposome [37].
composition, size, and charge of the vesicle. Liposomes with mod-
ified surfaces have also been developed using several molecules ADVANTAGES DISADVANTAGES
such as glycolipids or sialic acid [3,33], and modified and unmod- Increase in hydrophilicity PEG dilemma
ified dextrans [34], which afford stealth liposomal properties. A Increase in aqueous solubility Poor cellular uptake
breakthrough in long-circulating liposome production occurred Increase in hydrodynamic volume Ineffective endo/lysosomal escape
when the synthetic polymer poly-(ethylene glycol) (PEG) was Reduction in protein binding ABC phenomenon (multiple injection)
Reduction in RES uptake Obstructed interaction between
incorporated into liposome composition. Consequently, extended
ligands and target
blood-circulation of liposome was detected when the PEG was Disturbed drug release from carriers
presented on the surface of the liposomal carrier. Because of Increased normal tissue exposure
the presence of PEG, mononuclear phagocyte system uptake was
reduced and since they are “invisible” to the bodyś defense system
(reticuloendothelial system (RES)), sterically stabilized liposomes Different strategies for overcoming the disadvantages of PEGy-
are often called stealth liposomes [9,35]. Furthermore, PEG reduces lated liposome have been implemented. To enhance the cellular
the particles’ aggregation by steric stabilization; therefore, the uptake of PEGylated liposome, the ligands (e.g. proteins, vitamins,
PEGylated liposomes’ stability is improved during storage and peptides, antibodies and antibody fragments, nucleic acids etc.) for
application [36]. Although PEGylated liposome exhibits many receptors on the surface of targeted cells on PEGylated carriers can
advantages over earlier varieties, a dilemma (Table 1) regarding be displayed. So-called “active targeting” improves the selectiv-
using PEG for liposome coating appeared, associated with the dif- ity, binding and uptake of the carriers by targeted cells [38,39].
ficulty of cellular absorption of drugs and subsequent endosomal Endo/lysosomal escape of PEGylated carriers can be improved by
escape [37]. de-PEGylation. Intracellular environments with a low pH in endo-
somes/lysosomes (pH-responsive polymeric carriers) [37,40–43]
4 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984
Fig. 3. Liposome as a drug delivery system and routes of drug administration to the human body.
or the use of enzyme matrix metalloproteinase [44–46] can trigger
PEG cleavage.
Accelerated blood clearance (ABC) phenomenon, unexpected
pharmacokinetic change resulted usually from a second dose of
conventional PEGylated liposomes or after several time repeated
injections of PEGylated liposomes was detected [47–49]. To miti-
gate the induction of this phenomenon, different strategies were
proposed; the administration regimen change [50], reduction in
density of PEG on liposome surface [51], alternative polymers
usage such as poly(N-vinyl-2-pyrrolidone (PVP) [52] and cleavable
PEGylation. The most promising alternative is cleavable PEGylation
[37]. PEGylated long-circulating antibody liposomes should not be
overmodified, the quantity of attached antibodies should not com-
promise the longevity too much, to provide unhindered interaction
with target cells [53].
Active molecules can be delivered to the site of action, and at
present, several formulations of liposome are in clinical use. Drug
administration with liposome can be achieved using many routes
(Fig. 3) (intravenous, oral inhalation, local application, and ocular)
and these can be used for the treatment of various diseases [54].
Drugs and other substances can be entrapped either in the internal
aqueous phase of the liposome or in the lipid bilayers, depending
on their hydrophilicity [14].
Liposomes, however, suffer from problems of storage stabil-
ity caused by particle aggregation or fusion and PLs degradation,
hydrolysis or oxidation [55,56]. These disadvantages can not only
be eliminated, but also extended drug delivery route to oral and
inhalable administration can be achieved with the preparation of
a solid dry free-flow powder called proliposome [57–59]. They can
form liposomal structures via self-assembly in water.
3.1. Commercially available formulations of liposome
Till now many liposomal formulations have been developed for
a variety of drugs [8,9,60]. Ambisome® was the first liposomal drug
to enter the market three decades after the discovery of liposome
[61,62]. Around twenty liposome-based drugs are now approved
for clinical use (Table 2) and almost sixty products are in clinical
stages of development [60]. Liposome nanotherapeutics for cancer
treatment have been on the market since the 1990 s, and a variety
of liposomal drugs for the treatment of other diseases are in various
stages of clinical development (Table 2) [63].
Generally, four basic stages are involved in all methods of
liposome preparation; drying down of a mixture of lipids from
an organic solvent, dispersion of lipids in aqueous media, sep-
aration and purification of formed liposomes, and analyzing
the final product [3]. Nowadays, novel liposome technologies
for delivery of therapeutics such as Stealth liposome technol-
ogy, Non-PEGylated liposome technology, DepoFoamTM liposome
technology and Lysolipid thermally sensitive Liposome (LTSL) tech-
nology are used [8], and by changing the lipid bilayer composition,
size, and charge of the vesicle, long-circulating liposome with con-
trolled and gradual release of active substances can be synthesized
(Table 2).
For the purpose of different agents target delivery at a specific
place in the body (e.g. anticancer agent delivery to the tumors),
functionalization of liposome with a suitable ligand, (i.e. peptides,
antibodies, aptamers, small molecules, etc.) should be performed
with so called ligand-targeted liposome or targeted liposome.
Targeted liposomes do not affect healthy tissues, while the conven-
tional approach for e.g. cancer therapy results in low accumulation
of anticancer agents at the affected place with associated off-target
effects [64]. The main strategies for the therapeutic agent delivery
at affected site involve two approach: passive targeting (PEGylated
liposome with enhanced permeability and retention effect) and
active targeting (functionalized liposome with targeting ligands)
[64–66].
The cell penetrating peptide (CPP) assist delivery of therapeu-
tic agents using liposome as a nanocarriers can be performed: a)
through covalent conjugation of the drug molecules with a CPP, b)
 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984 5

Table 2
Clinically used liposome-based products, their active components, therapeutic applications, structure of lipid bilayer and preparation technology used [8,24].

Therapeutic application Liposomal preparation Active compound Method of preparation

(brand name) (lipid bilayer)

Cancer therapy DaunoXome® DAUNORUBICIN (DSPC and CH) Conventional technology

DepoCyt® CYTARABINE DepoFoam technology
(DOPC, triolein, DPPG and CH)
Doxil® DOXORUBICIN (PC and CH) Stealth liposome technology
EvacetTM DOXORUBICIN Conventional technology
(egg PC and CH)
Lipodox® DOXORUBICIN (HSPC, DSPE-MPEG-2000 and CH) Stealth liposome technology
Marqibo® VINCRISTINE (sphingomyelin and CH) Conventional technology
Mepact® MIFAMURTIDE Non-PEGylated liposome technology
MTP-PE (POPC and OOPS)
Myocet® DOXORUBICIN Non-PEGylated liposome technology
(egg PC and CH)
OnivydeTM IRINOTECAN Stealth liposome technology
(DSPC, CH and DSPE-MPEG-2000)
®
Fungal infection Abelcet AMPHOTERICIN B (DMPC and DMPG) Non-PEGylated liposome technology
AmBisome® AMPHOTERICIN B (HSPC, CH and DSPG) Conventional technology
Amphocil® AMPHOTERICIN B (sodium cholesteryl sulphate) Non-PEGylated liposome technology
Amphotec® AMPHOTERICIN B (cholesteryl sulfate) Non-PEGylated liposome technology
Analgetics DepoDurTM MORPHINE SULFATE (DOPC, CH, DPPG, tricaprylin, DepoFoam technology
and triolein)
Exparel® BUPIVACAINE DepoFoam technology
(DPPG, tricaprylin and DEPC)
Viral vaccines Epaxal® HEPATITIS A VACCINE (DOPC and DOPE) Detergent removal method
Inflexal® V INACTIVATED HEMAGLUTININE OF INFLUENZA Detergent removal method
VIRUS STRAINS A and B (DOPC and DOPE)
Photodynamic therapy Visudyne® VERTEPORFIN (egg PG and 1-␣-DMPC) Conventional technology

PC - phosphatidylcholine, CH - cholesterol, DSPC - distearoylphosphatidylcholine, DOPC - dioleoyl phosphatidylcholine, DPPG - dipalmitoyl phosphatidylglycerol, PS - phos-
phatidylserine, DSPE-MPEG-2000−1,2-distearoyl-phosphatidyl ethanolamine-methyl-polyethylene glycol conjugate-2000, DMPC - L-␣-dimyristoylphosphatidylcholine,
DMPG - l-␣-dimyristoylphosphatidylglycerol, DSPG – distearoyl phosphatidylglycerol, DEPC - 1,2-dierucoyl-sn-glycero-3-phosphatidylcholine, DOPE - 1,2-dioleoyl-
sn-glycero-3-phosphoethanolamine, POPC - 1-palmitoyl-2-oleoylphosphatidylcholine, OOPS - 1,2-Dioleoyl-sn-glycero-3-phospho-l-serine. HSPC - hydrogenated soy
phosphatidylcholine, DSPE-MPEG-2000−1,2-distearoyl-phosphatidyl ethanolamine-methyl-polyethylene glycol conjugate-2000.

encapsulation of drugs into CPP attached liposome, and c) by phys- equilibration method used for drug loading strongly depends on
ical adsorption of drugs with CPPs using electrostatic complexation lyophilization. Producing a liposomal drug kit product via the pas-
method [64,67]. With functionalization of liposome using targeting sive equilibration method could be performed by coupling a stable,
ligands better transport of liposome in cancer cells is provided [68], lyophilized empty liposome system to hydrophilic drug loading
nonspecific distribution to undesired tissues is decreased [69] and [72]. Mechanical dispersion methods include lipid film hydra-
high specificity and targeting efficiency are reached [64]. tion, microemulsification, sonication, French pressure cell method,
membrane extrusion, dried reconstituted vesicles and the freeze-
thawed method. Solvent dispersion methods include ethanol
4. Methods of drug loading in liposome preparation
injection method, ether infusion method, double emulsification
and reverse-phase evaporation [73]. Lipid-soluble, water-soluble
One of the critical issues in liposome preparation is loading
and amphiphilic materials may be encapsulated in liposome. This
drugs (hydrophilic or hydrophobic). In a multistage preparation,
is a way to decrease their side effects and toxicity [74].
it is important to know when and at what stage the drug is incor-
Using the filming-rehydration method, for example, multifunc-
porated into the system. Methods of liposome preparation can be
tional composite liposomes containing Laurel essential oil (LEO)
in general divided into two major groups; the active loading tech-
and silver nanoparticles (AgNPs) (Lip-LEO-AgNPs) were prepared.
nique with proliposome and lyophilization, where liposomes are
Later they were mixed with chitosan solution to cast on a polyethy-
obtained by transfer of PLs from an organic phase into an aqueous
lene (PE) film (PC-Lip/LEO/AgNPs) for pork preservation [74].
phase, and passive loading techniques with mechanical disper-
Instability of liposomes in the aqueous state during long-term
sion, solvent dispersion and the detergent removal method with
storage may affect the possibility of using them as drug delivery sys-
dialysis where lipid films are first deposited on a substrate and
tems. Liposomes, stored as aqueous dispersions, are instable since
subsequently hydrated to give liposomes. Active loading refers to
the encapsulated drugs tend to leak out of the bilayer structure
methods of incorporating drugs after liposome preparation. Usu-
and the liposomes might aggregate. Therefore, it is generally nec-
ally, these are gradient loading techniques involving buffer or
essary to use them within the first few months of preparation [75].
ammonium sulfate gradients. Passive loading, or passive aqueous
To overcome this problem lyophilization, as a drying technique
capture, is what happens for all techniques where a drug is captured
for thermolabile pharmaceuticals, should be used [75,76]. A typ-
during liposome formation process. Active loading refers to meth-
ical freeze-drying process consists of three main phases: freezing,
ods of incorporating drugs after liposome preparation. Usually,
primary drying and secondary drying, during which stresses of lipo-
these are gradient loading techniques involving buffer or ammo-
somal dispersions may occur [77]. To stabilize the liposomes during
nium sulfate gradients. Trans-membrane gradient is a driving force
lyophilization process different excipients (e.g. sugars, usually dis-
for active loading of a weak base (e.g. ammonium sulfate) into lipo-
accharides) or adjuvants (e.g. organic solvents, such as tert-butyl
somes and using this approaches, high EE can be obtained. For
alcohol) should be used [77].
appropriate loading, a weak base with a pKa≤11 and log P value
When the liposomes are formed on the basis of W1/O/W2 emul-
ranging from −2.5 to 2.0 should be used [70,71].
sions, the organic solvents are usually removed by evaporation at
Lyophilization has a significant effect on some sorts (e.g.
high temperature [78]. The disadvantage of using evaporation to
cholesterol-free, pegylated (CF-PEG)) of liposomes. The passive
6 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984

Table 3
Advantages and disadvantages of using conventional techniques for liposome formation.

Method Advantages Disadvantages Types of vesicles Reference

Reverse-phase evaporation
technique
Injection techniques
Electroformation methods
Microfluidic method
(1. Micro hydrodynamic
focusing (MHF), 2.
Microfluidic droplets (MD),
3. Pulsed jet flow
microfluidics (PJM))
Thin-film hydration (Bangham
method)
Detergent depletion
Membrane extrusion
Heating method
simple process,
suitable EE
simple process
homogeneous size distribution
good particle size control
simple process,
straightforward approach
good particle size control,
simple process,
homogenous product
simple and fast process,
good particle size control,
non contamination (e.g.
organic solvent)
simple and fast process,
non contamination (e.g.
organic solvent),
no sterilisation is needed
use of large quantity of organic MLVs, LUVs [19,89,90,91]
solvent (unsuitable for peptides
encapsulation),
time consuming method,
sterilisation is needed
difficult removal of organic solvent, SMVs, SUVs [92,93,94]
time consuming method,
possible nozzle blockage (ether
system),
sterilisation is needed
high cost of electrodes (mainly for GUVs [95,96,97]
platinum and ITO electrodes),
buffer with ionic low strength (a
maximum of 50 mM)
difficult removal of organic solvent, SUVs, LUVs (for MHF), [98,99,100,101,102]
unsuitable for bulk production GUVs (for MD),
GUVs (for PJM)
Novel methods for liposome
preparation
low EE (specially for water soluble MLVs [31,103]
active agents),
difficult removal of organic solvent,
small scale production,
large vesicles without particle size
control,
time consuming method,
sterilisation is needed
difficult removal of organic solvent, MLVs, LUVs [104,105,106]
detergent residue,
time consuming method,
poor EE,
sterilisation is needed
drawbacks in large-scale MLVs [107,108,109]
processing,
possibility of clogging the pores
high temperature MLVs, SUV [24,110,111,112]

SMVs - small multilamellar vesicles.

GUVs - giant unilamellar vesicles (size >1000 nm) [24].

remove solvent is in possible leakage of the inner aqueous solution with improved morphologies and minimal residual solvent. Dense
as well as agents by diffusion through the fluid oil phase. Fur- gas technology has become an alternative production technique
thermore, the evaporation process might break down the double that is more environmentally acceptable and economical than con-
emulsions and lead to drug loss [79]. ventional methods.

5. Conventional techniques for liposome preparation
Different conventional techniques of liposome formation,
such as the reverse-phase evaporation technique, injection tech-
niques, hydration methods, electroformation methods, microflu-
idic method, thin-film hydration (Bangham method), freeze drying
of double emulsions, membrane extrusion, detergent depletion,
heating method etc. are well documented [3,17,80–86]. Most of
them have drawbacks, such as complex procedures, working under
harsh process conditions (denaturation of active components may
occur) and poor drug EE [87]. Another drawback is the use of organic
solvents, since they have to be removed when liposomes are used
in pharmaceutical applications, namely, to produce pharmaceuti-
cals, besides safety rules also strict regulatory requirements must
be followed. This leads to an increase in production costs because of
the need for additional purification and waste disposal steps [88].
Advantages and disadvantages of using conventional techniques
for liposome formation are depicted in Table 3.
Therefore, there is incentive for the use of SCFs technologies as
a technological platform for the production of liposomal systems
5.1. Tools for size reduction and uniform size distribution of
liposome
Particle size of liposome is a very important parameter for their
application in medical and other in vivo applications, since it affects
different properties such as stability, encapsulation efficiency, drug
release, mucoadhesion and the cellular uptake of liposome [113].
Therefore, an important step in liposome preparation is also prepa-
ration of uniform-size distributed liposomes in a reproducible
manner. Liposomes with diameters >0.1 ␮m can be, in compari-
son to smaller ones, opsonized more rapidly and to a greater extent,
which leads to more rapid removal from the body by the reticuloen-
dothelial system [109]. Also, biodistribution of liposome is related
to particle size [114].
The initial hetero-dispersed suspensions of liposomes can be
reduced in size and size distribution by several different meth-
ods. One of the most commonly used methods, for size reduction
and distribution, which is suitable also for large-scale production,
is homogenization. During the process, hetero-dispersed liposome
preparation is cycled pumped under high pressure through a small
 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984 7

Table 4
Advantages and disadvantages of different size-processing methods for uniform particle size liposome.

ADVANTAGES DISADVANTAGES

HOMOGENIZATION suitable for large-scale production
SONICATIONORULTRASONIC IRRADIATION suitable for SUVs preparation
EXTRUSION several membrane/filter pore sizes are
available for producing liposomes in different
selected size ranges
quite narrow distribution of the liposome due
to several times cycling of the material
through the selected-size membrane/filter
liposome size distribution is quite broad and variable
possible metal and oil contamination
limited processing capacity
peroxidative damage to the lipids due to the local
overheating during the process
possible contamination with titanium
limitation in large-scale production
membrane/filter clogging
membrane/filter fragility
slow flow through the membrane/filter

reaction tank, until a desired average size of liposome is achieved.
Sonication or ultrasonic irradiation is another method for reduc-
ing the size of liposome because of the shear forces present during
the process. Another size-processing method for uniform liposome
preparation is extrusion through uniform pore-size membranes.
General advantages and disadvantages of different size-
processing methods for uniform particle size liposome formation
are depicted in Table 4.
In a study by Ong et al. [109], evaluation of different
size-processing methods (extrusion, ultrasonication, freeze-thaw
sonication (FTS), sonication and homogenization) to obtain nano-
size liposome was performed. The extrusion method was found to
be the most efficient, followed by FTS, ultrasonication, sonication
and homogenization. With increase in the flow rate of the extrusion
process, decrease in the size of extruded liposome was deter-
mined. Furthermore, the liposome size and distribution declined
with decrease in membrane pore size.
6. Supercritical fluids (SCFs) methods for liposome
preparation
Traditional methods of preparing liposome, such as thin film
hydration or ethanol injection, have many shortcomings, such as
poor monodispersion, poor stability, high residual organic solvent,
and side effects [115]. In addition, organic solvents used for conven-
tional liposome preparation may cause environmental pollution.
They may also become toxic and degrade the active ingredient,
presenting a possible risk for human health [116]. In the case of pro-
tein drugs, detergents or organic solvents can cause denaturation
of proteins and affect the membrane properties [117].
Supercritical fluids assisted techniques, such as supercriti-
cal anti-solvent (SAS), rapid expansion of supercritical solutions
process (RESS), supercritical assisted atomization (SAA), super-
critical fluid extraction (SFE) [118–120] or supercritical reverse
phase evaporation (SCRPE), particles from gas saturated solutions
(PGSS), depressurization of an expanded liquid organic solution-
suspension (DELOS) [17,121–125] and supercritical assisted
liposome formation (SuperLip) [126], are being developed as
successful alternatives to traditional methods for liposome pro-
duction [127,128]. They can be classified as green technologies.
The most widely used supercritical gas is carbon dioxide, since it
is non-flammable, non-toxic, non-corrosive, inexpensive, environ-
mentally acceptable and has easily manageable critical parameters
of 31.1 ◦ C and 7.38 MPa [129]. It is also suitable for use with thermo-
sensitive materials [130], and a low energy input is required for SC
CO2 processing.
Liposome, produced with the SC CO2 method have enhanced
intactness, sphericity and uniformity compared to liposome, pro-
duced by the thin film hydration method. Dense phase CO2 enables
the processing of phospholipid (PL) aggregates into nano/micro
particles. Their characteristics can be controlled via tuning the
processing parameters [131]. The rate of decompression and the
opening diameter of the nozzle control the size of the resulting
liposomes.
6.1. Depressurization of an expanded liquid organic
solution-suspension (DELOS) method
For DELOS process, the substances are first dissolved in suitable
organic solvent and then the solution is mixed with SC CO2 in a
vessel at desired temperature and pressure. Then the mixture is
depressurized through a nozzle into a vessel to form liposomes.
Therefore, the SC CO2 is used as co-solvent to the organic solvent.
The main advantage of this technique in comparison to PGSS is that
the thermo-sensitive material can be handled to prepare fine par-
ticles without melting it and DELOS process requires mild working
pressure (e.g. 10 MPa 35 ◦ C), which is a very important param-
eter from the economical point of view, especially for industrial
application [132,133]. A possible mechanism of liposome forma-
tion via the SC CO2 method (DELOS) is illustrated in Fig. 4. Four
stages of the process exist. In the first stage, PL molecules are
present primarily in the form of bilayer or curvature organized
spontaneously in the aqueous medium at ambient conditions. In
the second stage, CO2 is rapidly solubilized in the aqueous phase
during pressurization. Dissociation equilibrium results with a rela-
tively small amount of CO2 in carbonic acid form and the remaining
amount in the unhydrated state [134]. In the third stage or depres-
surization, CO2 molecules are rapidly released from PL bilayers to
temporarily disrupt them into highly dispersed PLs. In the fourth
stage after formation of the instantaneous solution, temporarily
separated PLs and CH reorganize rapidly, as a result of hydrophobic
and van der Waals interactions and finally pack into drug-loaded
liposome [131].
Lutein-loaded liposomes were prepared using the DELOS
method [135]. The expansion of CO2 -PL/lutein suspension which
depressurized from high pressure to ambient conditions resulted
in liposome formation. Characterization via several techniques
showed that liposomes with superior characteristics were formed.
Liposomes prepared with the SC CO2 method had a particle size
of 147.6 ± 1.9 nm – 195.4 ± 2.3 nm and exhibited 87.1 % of unil-
amellar, 8.7 % of bivesicular and 4.2 % of bilamellar spherical vesicles
and EE of 56.7 ± 0.7 %–97.0 ± 0.8 %. At higher pressure (20–30 MPa)
and depressurization rate (9–20 MPa/min) a higher encapsulation
of lutein was obtained. Also, lutein-to-lipid ratio influenced the
morphology of vesicles along with size distribution and EE. [135].
The bilayers vesicles formation most likely occurred because of the
depressurization and dispersion of the two nearby domains of PLs.
The reorganization of dispersed internal molecules of PLs formed
an inner bilayer which is surrounded by the outer one formed by
external LPs [135].
Utilization of liposome as an encapsulation medium for antho-
cyanin via DELOS method may provide unique features to regulate
8 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984

Fig. 4. Liposome formation by the DELOS method - Schematic illustration of proposed mechanism where substances are first dissolved in suitable organic solvent and then
the solution is mixed with SC CO2 in a vessel at desired temperature and pressure. Then the mixture is depressurized through a nozzle into a vessel to form liposomes [131].

the characteristics for its protection, oral administration and

delivery purposes. Liposomes were formed as the CO2 -expanded
suspension was depressurized. Particle size of with SC CO2 pre-
pared anthocyanin-loaded liposome was 159 ± 0.2 nm and EE of
50.6 % at 30 MPa, 9 MPa/min and 50 ◦ C [136]. The anthocyanin
release from liposomes was slow in the simulated gastric fluid but
rapid in the simulated intestinal fluid, induced by the degradation
of the vesicles by pancreatin [136]. High concentration of added
anthocyanin leads to imperfect formation because of enhanced
anthocyanin-phosphate interactions and dividing the drug into the
bilayer. On the other hand, the increase in CH concentration has a
relatively small impact on the formation of liposomal bilayers and
shape but leads to a reduced surface charge of liposomes. Using the
DELOS method at 30 MPa and 50 ◦ C, mostly unilamellar, without
any multilamellar or multivesicular vesicles, were formed [136].
Again, in in vitro release study of melatonin liposome showed
that liposomes produced using SC CO2 were resistant to low pH
in simulated gastric conditions [137]. In a simulated intestinal
environment, enteric solubility of these liposomes, which is an Fig. 5. Liposome preparation using SAS method. The SCF is first pumped to the top
important property for controlled release of bioactive ingredients, of the high-pressure vessel until the system reaches a constant temperature and
pressure. Subsequently, active substance solution is sprayed as fine droplets into
was enhanced. Compared to those produced using the thin-film
above SCF bulk phase through an atomization nozzle. Liposomes are formed in a
hydration (TFH) method, SC CO2 -produced liposome toxicity was second step, when the hydration of the processed material is carried out.
much lower. This was confirmed by analysis of the organic solvent
residue in the liposomes using gas chromatography - mass spec-
trometry [137]. Additionally, when the liposomes were formulated because of many advantages. It enables lower residual solvent con-
using the DELOS method, the mean diameter of the liposomes was tent, is a relatively simple process, and is able to process molecules
66.19 nm and the polydispersity index (PdI) was 0.267, while when with poor solubility in the SCF, as the SCF is used as an anti-solvent
the TFH method was used, mean diameter of the liposomes was [139]. The SAS process has often been used for the precipitation
85.62 nm and the PdI was 0.382, but larger mean diameter devi- of compounds difficult to comminute, such as lipids. An organic
ations using the TFM method were detected. When the liposomes solution containing the solute (drug incorporated in liposomes) to
were formulated with the DELOS method, with increase in pres- be precipitated is placed in contact with a SCF, such as SC CO2 . SC
sure from 10 to 14 MPa, the particle size of the liposomes decreased CO2 acts as an anti-solvent for the solute but is completely miscible
from 88.74 to 66.19 nm. With further increase in pressure, the mean with the organic solvent. When the SAS process is used, in the final
diameter of formed liposomes increased. Therefore, for easier con- obtained liposomes no traces of organic solvents are present com-
trol over dimensions and distribution of liposomes, the SCF method pared to liposomes obtained by conventional methods. Liposomes
is more convenient [137]. are formed in a second step, when the hydration of the processed
Overall, this SC CO2 assisted method was demonstrated to material is carried out [140]. They are hydrated at ambient condi-
be superior for the preparation of liposomes over the traditional tions by an aqueous solution (Fig. 5). The SAS process appears to
TFH method, resulting in small size, relatively high uniformity be an efficient and environmentally friendly process for producing
and enhanced stability of spherical shape, while offering modified liposomes.
physicochemical properties of bilayer membranes and the possi- Vitamin D3 (VD3) proliposomes (VDP) were prepared to make
bility of reducing the usage of sterols [138]. VD3-liposomes (VDL) using SAS via hydration of the proliposomes.
VDP can be first produced by SAS and then the liposomes can be
6.2. Supercritical anti-solvent (SAS) method simply formed via hydrating [141,142].
1,2-Distearoyl-sn-glycero-3-phosphocholine(polyethylene gly-
The most commonly reported methods for proliposome prepa- col) (DSPC-PEG) liposomes loaded with albendazole (ABZ) using
ration are SAS methods. This is the most attractive technique SAS were also prepared. ABZ is a colorless, non-ionizable, lipophilic,
 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984
Fig. 6. Liposome preparation using RESS method. RESS process is consisting of two
steps: (a) dissolving the solid substance and carrier in a SCF and (b) formation of
particles due to supersaturation.
low molecular weight crystalline compound which is insoluble in
water and has promising anticancer activity when formulated to
increase its cellular uptake. The diameter of unilamellar and spher-
ical liposomes was 167.2 ± 5.3 nm and a controlled release effect on
ABZ release profile was observed The experiments were performed
at 37 ◦ C, 25 MPa. [143].
Using the SAS technique, hydrophilic and hydrophobic com-
pounds can be entrapped in lipid vesicles. In this process, the
compounds to be encapsulated do not need to be dissolved in SC
CO2 .
The SAS process was upgraded to what is called the continuous
anti-solvent (CAS) process. In the SAS process, micronized PLs react
spontaneously upon contact with air [144] before the hydration
step. CAS affords a solution to this problem because in this process,
micronization and hydration are performed in the same autoclave
under pressure [119].
6.3. Rapid expansion of supercritical solutions (RESS) method
RESS process is consisting of two steps; (a) dissolving the solid
substance in a SCF and (b) formation of particles due to supersatura-
tion. In the RESS process, at first SCF is pumped at desired pressure
and temperature to extractor. The SCF percolates and dissolves the
solid substance(s) in the extractor and then the resulted solution is
depressurized through a heated nozzle in a low-pressure chamber
(Fig. 6) [2]. RESS process is simple, effective when single nozzle is
used and it minimizes the use of organic solvent and reuses the SCF
in continuous process. The main drawback is poor solubility of most
of the pharmaceutical materials (e.g. polymer) in SC CO2 , therefore
large amount of fluid is required, consequently the cost of liposome
production using RESS method is increased. Additional, particles
may aggregate and nozzle block because of cooling during rapid
expansion of the supercritical solution [145,146]. Drug-loaded lipo-
some can also be prepared via RESS; the operating conditions of
RESS have been found to strongly affect the properties of the pre-
pared liposomes. The size, uniformity, and EE of the liposomes can
be varied by changing the pre-expansion pressure, flow rate, and
the position of the nozzle in the reactor. Liposomal microcapsules
were produced using SC CO2 without using any organic solvent,
CH, or external energy for pumping and mixing the solution. The
liposomes’ sizes became uniform when the pressure was increased
from 12.4 MPa to 17.2 MPa [147].
Vitamin C (VC) liposomes were prepared by RESS; EE of the pre-
pared VC liposomes was 75.38 ± 1.03 %. The particle size (spherical
shape), PdI and Zeta-potential of the prepared samples was 270.4 ±
9
Fig. 7. SuperLip process [154]. SuperLip process is consisting of two steps: first,
water droplets produced by atomization inside a high-pressure vessel, filled with an
expanded liquid mixture formed by PLs/ethanol/CO2 , containing drugs are obtained
and in the second step, a lipidic layer is formed around them.
5.2 nm, 0.254 ± 0.010 and −41.7 ± 0.9 mv. Liposome were prepared
at following condition; 25 MPa, 48 ◦ C, feed ratio of VC against PC
of 0.25, while the flow rate of the sprayed mixture was about 15
g/min [148].
Zhang et al. prepared sirolimus liposomes using RESS and the
effects of temperature, pressure, and equilibrium time on the aver-
age particle size and envelope rate of the liposomes were discussed
in detail [149]. Wen et al. reported a modified technique of RESS
to prepare essential oil liposomes [150,151]. The entrapment effi-
ciency of the prepared essential oil liposomes was found to be 82.18
% under optimum conditions (pressure of 30 MPa, temperature of
65 ◦ C and ethanol mole fraction in SC CO2 of 15 %) with an average
particle size of 173 nm (spherical shape). Xu et al. successfully syn-
thesized nanoscaled CoQ10 liposomes (20−40 nm, spherical shape)
using RESS under different pressures. Surprisingly, the trapping
efficiency of the liposomes can be up to 90 % when the enduring
pressure was 20–30 MPa [152]. Melatonin-loaded liposomes with
uniform size distribution and an average diameter of 66 nm were
also successfully prepared using RESS [153]. The EE of the lipo-
somes could reach 82.2 % under optimal conditions (14 MPa and
40 ◦ C), and only 1.03 wt% of ethanol (residual organic solvent) was
detected in the products, eliminating some potential dangers for
practical applications [153].
Frederiksen et al. reported the preparation of liposomes con-
taining a water-soluble marker using the RESS method (7% absolute
ethanol and carbon dioxide at 25 MPa and 60 ◦ C). The average size
of the liposomes was about 200 nm, and the total amount of ethanol
used to obtain an EE of 20 % was 15-fold reduced compared to the
ethanol injection method [125].
6.4. Supercritical assisted liposome formation (SuperLip) method
SuperLip (Fig. 7) consists of an inversion of the traditional lipo-
some production steps: first, water droplets containing drugs are
10 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984
obtained and in the second step, a lipidic layer is formed around
them. Water droplets are produced by atomization inside a high-
pressure vessel, filled with an expanded liquid mixture formed by
PLs/ethanol/CO2 . These droplets are rapidly surrounded by a lipid
layer, forming a w/CO2 emulsion, and liposomes (w/w emulsion)
are formed when they fall in the water pool located at the bot-
tom of the vessel [126]. The vesicles obtained are nanometric and
sub-micrometric and may entrap proteins, antibodies, antibiotics,
essential oils, agricultural additives, antioxidants, dyes and hol-
low gold nanoparticles [154]. This process offers the possibility
of producing liposomes with a complex architecture of the lipid
membrane. The difficulties experienced using the classical lipo-
some formation processes, mainly in reproducibility and EE, can
be overcome with this method [155].
SuperLip was used for the liposome production to deliver the
ophthalmic antibiotics ampicillin and ofloxacin, antibacterial drug
used to stop ocular post-surgery infections. The process parameters
for liposome production were: water injector diameter of 80 ␮m,
operative pressure at 10 MPa and temperature at 40 ◦ C, CO2 /ethanol
ratio (gas to liquid ratio (GLR)) was 2.4 w/w, CO2 flow rate was
6.5 g/min, and drug/lipid ratio was 1% w/w. EE for ofloxacin and
ampicillin were up to 97 % and 99 %, respectively. Mean diameters
of the liposomes, which were ULV, were in the range from 280 ±
104 nm to 1.76 ± 0.79 ␮m. The liposomes were stable for at least 3
months, with negligible drug leakage [156].
A polyphenol-rich aqueous extract from olive pomace was
encapsulated in liposomes using SuperLip process. Preservation of
these temperature sensitive compounds demands mild operative
conditions which could be achieved using this process. The mean
diameters of liposomes with spherical shape and a slightly smooth
surface produced at 13 MPa was smaller than 265 nm, and for lipo-
somes produced at 17 MPa, was 168 nm. EEs were about six times
larger than using conventional methods and were up to 58 % [157].
The amphiphilic antioxidant eugenol was entrapped in the inner
core and in the lipophilic double layer of liposomes using SuperLip
for their preparation. On the other hand, liposomes with lipophilic
(␣-lipoic acid) antioxidant in the lipidic layer were also produced
with the same technique. EEs were about 94 % and 68 % respectively,
and their antioxidant power was preserved after processing. Lipo-
somes of nanometric dimensions with spherical shape and smooth
surface were produced and their mean diameters ranged between
255 ± 122 nm and 230 ± 96 nm. The operation conditions were:
10 MPa, 35 ◦ C, ethanol solution flow rate was 3.5 mL/min, CO2 flow
rate was 6.5 g/min (GLR = 2.4 (w/w) and water flow rate was 10
mL/min [158].
To manufacture liposomes from PLs using SCF technology, their
dispersibility was determined. 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 (ULV), while using
the thin film method it was 153.6 ± 4.5 nm and 131.3 ± 3.4 nm
(ULV), respectively. SCF mediated liposomes were better in parti-
cle size and morphology when compared with those produced with
the thin film method. They were also more stable over a period of
3 months compared to those produced with the thin film method.
This green alternative SCF method of liposome preparation is less
laborious, saves time, and is a low energy process [159].
Spherical PC/CH and PC/PE liposomes were produced with nano-
metric mean diameters down to 200 nm and theophylline was
encapsulated with EE up to 98 % at operation parameters: ethanolic
solution flow rate of 3.5 mL/min (2.76 g/min), GLR = 2.4 (w/w), 10
MPa, 40 ◦ C and water flow rate of 2.14 mL/min. Liposome size dis-
tribution, vesicles stability and drug EE were not affected by lipid
bilayer composition [155].
Campardelli et al. [160] studied the possibility of producing
liposomes with a different composition of the bilayer membrane
(composed of PC or a mixture of PC/PG) using the SuperLip process.
Spherical PC liposomes of different sizes and distribution ranging
between 250 ± 58 nm and 330 ± 82 nm with wrinkled surfaces
were successfully produced at 15 MPa and 40 ◦ C. When using a
PG/PC mixture, larger liposomes were produced (280 ± 70 nm and
350 ± 101 nm). Both PC and PC/PG liposomes were stable over
one month (zeta potential ranging between −20 mV and −30 mV)
and very high EE for BSA (92–98 %) were obtained. The authors also
found that pressure and temperature influenced the mean diameter
of liposome. The higher the pressure and the lower the tempera-
ture (i.e. the higher the expanded liquid density), the smaller the
mean diameter of liposomes.
A novel approach to include lipophilic molecules in liposomes
using emulsions was developed by Trucillo et al. [161]. Two dif-
ferent strategies for the encapsulation of hydrophobic bioactives
within nanoliposomes were attempted. Lipophilic antioxidants
were entrapped in liposomes using emulsions: a drug was dissolved
in the oil phase of an oil in water emulsion, then the aqueous emul-
sion was entrapped in the liposomes’ inner core using SuperLip.
The O/W emulsion method successfully preserved the antioxidant
activity of the entrapped molecules. High preservation of antiox-
idant power using emulsion loaded liposome was obtained. The
reduction of antioxidant power was about 22 % for lipidic layer
entrapment but only 2% using the O/W method.
6.5. Other methods
A GAS process for liposome preparation is usually used instead
of RESS technology when the substances are poorly soluble in SCFs.
GAS process is a batch process where the precipitator is partially
filled with the solution of solute and SCF is used as an anti-solvent
for precipitating a solute (active compound), which is insoluble in
SCFs, and that has been dissolved in an organic solvent. The parti-
cles precipitate as the gas concentration in the solution increases
with pressure. A clear disadvantage of this technique is the lack of
control on the particle formation [162]. Water soluble compounds
of Cordyceps sinensis CS1197 were encapsulated in nano-liposomes
with a mean diameter of 72.0 ± 2.0 nm using the GAS method,
and much smaller liposomes were formed, compared to the con-
ventional method. GAS mediated liposome production unit can be
operated in fed-batch mode. Reproducibility of the encapsulation
of both hydrophilic and hydrophobic active compounds is ensured
[163].
Nano-liposomes were designed using the GAS method. The GAS
method was compared with Bangham’s method in terms of the
mean diameter, coefficient of uniformity, morphology, stability
index and actual energy required for liposome formation. Liposome
production using the GAS method under optimized formulation
conditions (17 MPa, 50 ◦ C, 1.5 % Tween 80 and a depressurization
rate of 2.5 MPa/min) yielded nano-liposomes exhibiting a coeffi-
cient of uniformity of 1.10 ± 0.012 with a mean diameter of 63 ±
2 nm and with a stability of up to 3 months at different tempera-
tures. The GAS process requires 1.52 times less energy compared
to Bangham’s method to achieve nano-scale liposome production
[164].
SCRPE provides a one-step method for the preparation of LUV
liposomes with a high EE for both water-soluble and oil-soluble
substances, allowing aqueous liposome dispersions to be obtained
through emulsion formation by introducing a given amount
of water into a homogeneous mixture of SC CO2 /lipid/ethanol
[123,165]. Lipids, organic cosolvent and SCFs are combined in a
stirred reactor at a temperature above the lipid phase transition
temperature and an aqueous solution is then slowly added to the
reactor. Because of the release of SCFs, liposomes are formed.
Bile salt-mediated liposomes containing sodium glycocholate
were prepared using an improved supercritical reverse evaporation
(ISCRPE) method which fully eliminates the use of organic solvent
 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984
(ethanol). Formulated as notoginsenoside R1 (NGR1) carriers, they
can be used for oral administration to improve bioavailability [166].
The mean EE, particle size, and polydispersity index (PDI) of the
optimized liposomal formulation (NGR1@Liposomes) were 49.49
%, 308.3 nm, and 0.229 at optimal conditions 20 MPa and 60 ◦ C. For
ISCRPE, higher pressure contributed to the formation of smaller
vesicles, while a higher depressurization rate boosted the homo-
geneity of the vesicles. Pressure of at least 10 MPa is recommended
to achieve a spherical morphology [167].
Disadvantages of SCF-based operations involve mechanical,
chemical, and biological hazards. Because of the use of high pres-
sure when flammable and/or explosive fluids are used in the
process, mechanical hazards may appear. A possible leakage of
CO2 (if used in the process) presents a health issue and must be
prevented using an over dimensioned vent line, ensuring a good
dispersion of the gas in the outside atmosphere [139]. But most of
all, the high cost of the equipment is the major drawback using SC
CO2 for the liposome production.
Combined methods give additional advantages and are often
used for liposome production. High EE, good stability, low side
effects and low residual solvent are achieved when using a com-
bined method of thin film hydration and SC CO2 technology [168].
A liposomal hydrogel with embedded curcumin (Cur) for treat-
ment of infected wounds was prepared using a combined method
of thin-film evaporation and SC CO2 technique (TE-SC CO2 ) with
freeze-drying. Obtained thin lipid film was incubated at 40 ◦ C and
18 MPa for an hour in SC CO2 . The organic solvents were removed
after depressurization. The drug is released via phospholipase A2
hydrolysis of lecithin in liposome with spherical vesicular structure
and a size of 188 ± 12 nm [169].
With thin film rehydration TE-SC CO2 , Cur was loaded in
a bilayer of liposomes embedded in three-dimensional porous
chitosan/␤-GP thermosensitive gels to form thermosensitive
liposome- hydrogels. Sol at room temperature and gel at body
temperature can be used for injectable hydrogel which acts as a
drug carrier. In comparison to Cur-liposomes prepared using the
FH method, Cur-liposomes prepared with the TE-SC CO2 method
had small particle size, good monodispersity and were more stable
[170].
The same method was used for a novel liposome preparation
with photo-responsiveness for controlled drug release. Two
model drugs and a photo-responsive reversible switch, Cur and
4-butylazobenzene-4-hexyloxy-trimethyl-ammoniumtrifluoro-
acetate (BHA) were used in liposomes in which BHA was
encapsulated in polar groups of PLs in a bilayer of liposomes.
Spherical nanoparticles with a typical vesicle structure and good
monodispersion with an average size of about 140 ± 15 nm were
obtained when the thin lipid film formed was hydrated with
double distilled water and incubated in SC CO2 at 40 ◦ C and 20
MPa for 1 h. When the BHA liposomes were formed by traditional
film hydration, polydispersity was detected; the average liposome
sizes were 100 ± 20 nm and 830 ± 23 nm, respectively. [171].
Solution enhanced dispersion by SC CO2 (SEDS) has been proven
to be a prominent method for preparing proliposomes because of
its mild operation, product controllable and no need of auxiliary
materials [172]. With this method, berberine proliposomes with EE
of 90.3 % ± 4.9 % (at 37.8 ◦ C and 18.3 MPa) and nanoscale spherical
particles with mean diameter around 80 nm and neutral zeta poten-
tial were prepared to improve the oral bioavailability of berberine
[173].
Liposomes can be prepared using a modified supercritical pro-
cess via depressurization of the liquid phase. This supercritical
fluids assisted method is based on a modification of the ISCRPE
and DELOS methods, to combine the advantages of both techniques
by employing a PL water suspension similar to that in ISCRPE and
depressurization from the liquid phases in DELOS, for improved
11
liposome preparation. While increased pressure is favorable for the
formation of smaller vesicles, liposomes prepared at ≥10 MPa dis-
play a spherical and stable shape, while higher depressurization
rates resulted in enhanced uniformity [174].
SFE, followed by RESS and vacuum-driven cargo loading, was
used for vitamin C and E hydrophilic and lipophilic compound
encapsulation in integrated liposomes. Vitamin E acts as the pri-
mary antioxidant and the resultant vitamin E radical then reacts
with vitamin C to regenerate itself [175,176]. This is how they per-
form synergistic action on their antioxidant activity to scavenge
free radicals in human bodies. The average vesicle size was 951.02
nm with a zeta potential of −51.87 mV. Most of the liposomes
formed using the SCF method were LUVs and MVVs. The EE attained
was 32.97 % for vitamin C and 99.32 % for vitamin E [177].
The SCF process offers a sustainable and energy-saving tech-
nique for rapid and continuous production of integrated liposomes.
These have the capability to microencapsulate a versatile group of
bioactive compounds for food and pharmaceutical applications.
PL vesicles may also be prepared using high-pressure CO2 /water
systems. PL molecules might act as a surfactant at the high-pressure
CO2 and water interface and form a self-assembled membrane in
the water phase after decompression of the systems. PL vesicles
were efficiently formed in liquid (fluid)–liquid systems [178].
Depressurization of an expanded solution into aqueous media
process, developed by Meure et al. [121], is a dense gas technique
in which liposomes can be produced in a single-step process at
moderate temperature and pressure. The gas-expanded solution is
released through a nozzle into an aqueous medium [87]. The major
advantage of this process, compared to conventional methods, is
the reduction of the residual solvent content in the product to 2.2
% (v/v), which is significantly lower than those reported for con-
ventional processing [89,92,93,179]. Supercritical fluid extraction
(SFE) was used not only to remove the ethanol residue from lipo-
some suspensions prepared by ethanol injection but also to affect
their size and distribution leading of liposomes. Nanosomes with
a mean diameter of about 180 ± 40 nm with a good storage sta-
bility were obtained at 12 MPa and 38 ◦ C. This technology is easy
to scale up to an industrial scale and merge in one step the sol-
vent extraction, liposome size engineering and an excellent drug
encapsulation in a single operation unit [180].
6.6. Batch or continuous SCF processes for liposome preparation
Different SCF techniques for liposomes preparation, where a
SCF is used as a solvent (SCRPE, RESS), as a cosolvent (SuperLip,
DELOS), as an antisolvent (GAS, SAS, SEDS, ASES) or as a dispersing
agent (ISCRPE, PGSS) can take place in a batch and/or continu-
ous manner (Table 5). Long-term, continuous process saves costs,
energy and time, when it is properly implemented but on the other
hand it is more expensive than the batch process. However, when
comparing the production rate, batch has lower rate than the con-
tinuous one. The shut-down times in continuous process are rare,
whereas in a batch process they are often. Continuous operations
yield more consistent product quality, more predictable behavior
and a better opportunity to maintain and perfect a steady-state
process. Batch processing may also be more often required in phar-
maceutical industries, while good manufacturing practice (GMP)
is required. However, continuous flow is becoming a more viable
option for industrial application, due to advancements in design
and technology.
Since liposomes are most commonly used as a drug delivery sys-
tem, their production with SCFs techniques should be adapted to
GMP requirements and be implemented on an industrial scale using
single step continuous operation. In order to ensure GMP require-
ments, the equipment should be constituted of stainless steel with
a smooth wall, without edges, easily washable and the substrates
12 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984

Table 5
Different SCF techniques for liposomes preparation using batch or continuous operation.

Method Lipid/active ingredients Operation Particle size EE Reference

PC and CH/ MELATONIN batch process 88.74−66.19 nm (influenced / [137]

DELOS by pressure)
soy lecithin and CH/ batch process 160 ± 2 nm 52.2 ± 2.1 %. [131]
ANTHOCYANIN
POPC and CH/ FLUORESCEIN batch process 200 nm 20 % [125]
isothiocyanate-dextran
(FITC-DEXTRAN) or zinc
phthalocyanine tetrasulfonic
acid (TSZnPc)
HSPC, DSPE-MPEG-2000 and batch process 231.3 ± 6.74 nm (spherical 91.9 ± 3.45 % [181]
CH/ THYMOQUINONE vesicles)
SAS
soy lecithin S75 and CH/ semi- continuous 0.1–10 ␮m (spherical vesicles) 10−20% [120]
CALCEIN process
hydrogenated PC/ VITAMIN D3 batch process 200 nm (spherical shape) 100 % [142]
HSPC, DSPG and CH/ batch process 137 nm (uniform spherical 90 % [182]
AMPHOTERICIN B shape)
PC and CH/ ATRACTYLONE batch process 124 nm (double-layered 87.25 % [151]
hollow spheres)
Tween 61 and CH/ NON batch process 239−969 nm 28 % [183]
RESS
PL and CH/ SIROLIMUS batch process 150 nm 58 % [149]
HSPC, SPC, CH and batch process of 269.2 ± 2.8 nm (spherical, 79.2 ± 4.4 % [184]
DSPE-PEG-2000/ DOCETAXEL SUVs)
PC and CH/ COENZYME Q10 batch process 20 – 40 nm (spherical shape) 90 % (pressure influenced) [152]
soy lecithin and CH/ continuous 400−1200 nm (pressure 31.65 % [185]
d-(+)-GLUCOSE process influenced; LUVs and MVVs)
soybean PC/ BOVINE SERUM continuous process 130 ± 62–294 ± 144 nm 85−90% [126]
ALBUMIN
SuperLip
L-␣-PC/ FARNESOL, LIMONENE, continuous process 116 ± 32 nm - 230 ± 62 nm 74 % for farnesol, 87 % for [161]
LINALOOL (spherical shape) limonene and 54 % for
linalool
PC/CH and PC/PE/ continuous process 200 nm 98 % [155]
THEOPHYLLINE
L-␣-PC/ FLUORESCEIN or continuous process 100−300 nm (spherical shape) 99 % [186]
AMPICILLIN
L-␣-DPPC and L-␣-DOPC/ batch process (at low ethanol concentration - 40 % [187]
SCRPE d-(+)-GLUCOSE MLVs at higher ethanol
concentration LUVs)
L-␣-DPPC and CH/ batch process 0.1–1.2 ␮m (LUVs) 20 % [123,188]
d-(+)-GLUCOSE
DPPC, PEG-DSPE and CH/ batch process 100−2000 nm (spherical 70 % [189]
BOVINE SERUM ALBUMIN shape)
ISCRPE (improved different PLs/ d-(+)-GLUCOSE batch process LUVs 36 % [188]
SCRPE) L-␣-DPPC and chitosan/ batch process 500 nm (LUVs) 17 % [190]
d-(+)-GLUCOSE
SEDS HSPC and soya PC/ SILYMARIN semi-continuous 160.5 nm (LUVs) 91.4 % [191]
process
PGSS Soybean lecithin and CH/ batch process 1.4–25 ␮m (LUVs, MLVs and 6–14.5 % [192]
LAVANDIN ESSENTIAL OIL MVVs)
GAS PC and CH/ AMPHOTERICIN B batch process 3.5–10 ␮m (spherical shape) 20 % [193]
ASES PC, CH and Poloxamer 407/ batch process 2.7–9.4 ␮m (pH influenced) 60 – 100 % [88]
MICONAZOLE

and other components should be of GMP quality. According GMP,
the procedures should be standardized and monitored, each stage
of the preparation has to be validated and automated cleaning pro-
tocol is mandatory [194,195].
7. Conclusion
Several liposomal formulations are already on the market, and
new studies are being conducted in the use of advanced liposome
preparation technologies. Liposome preparation methods have sig-
nificant effects on their size, shape and lamellarity, which influence
the EE. Conventional methods of liposome preparation are not
suitable for achieving free-solvent liposomes and industrial-scale
production under current good manufacturing practice conditions
[18]. They also suffer from low EE of hydrophilic compounds, dif-
ficult removal of organic solvents, and poor control of particle size
distribution. Liposomes formulation using SCFs avoids the exten-
sive use of organic solvents (because of their toxicity, they represent
possible health risks), avoids the use of water, high operating
temperatures, and mechanical stresses that can degrade labile com-
pounds such as vitamins, enzymes, essential oils, and flavors, and
enables liposomes dry powder formulations. This green technol-
ogy can produce micro- and nano-sized liposomes with narrow
size distribution by altering critical conditions (i.e., temperature
and pressure). SCFs methods for liposome formulation also enable
high EE [17]. It has also been argued that SCFs assisted methods
can be scaled-up without much difficulty [17]. Indeed, the super-
critical extraction of liposomes is easy to scale up to an industrial
production and merge in one step the solvent extraction [180],
or ISCRPE has a scale-up potential [85] but at RESS scaling up of
the process is difficult, because of particle aggregation and noz-
zle blockage caused by cooling due to the rapid expansion of the
supercritical solution [145,146]. Batch and semi-batch technolo-
gies such as RESS, SAS, SAA etc. are quite complex to scale up due to
their discontinuous operations. Therefore, we cannot simply gen-
eralize this fact, but further evaluation of each process shall be
done. Several different SCFs assisted methods for liposome prepara-
tion have been developed. Because of the non-polar characteristics
 L. Maja, K. Željko and P. Mateja / J. of Supercritical Fluids 165 (2020) 104984
Table 6
Particle size and EE for liposomes prepared using different supercritical assisted
methods.
Method Particle size distribution (nm) EE (%)
DELOS very narrow (150−200) low (20–50)
SAS very wide (100–10000) medium (20−100)
RESS narrow (20−900) medium (20−90)
SuperLip wide (100–1700) high (60−100)
of the most commonly used SCF, SC CO2 , the EE of hydrophilic
compounds in liposomes is still low. A brief comparison between
different supercritical assisted methods for liposome formulation
in terms of particle size distribution and EE is given in Table 6.
The SuperLip method has proven to be an advanced method that
can circumvent the problem of low EE, since the water droplets
are first generated and then they are covered by PLs, leading to
the production of monodispersed, organic solvent free and high EE
hydrophilic drug liposomes. Another technique which also facili-
tates high EE is the SAS technique, followed by RESS. On the other
hand, DELOS results in the lowest EE, but with this method, very
narrow particle size distribution appears, which is not the case
when other SCFs methods are used.
Although the use of SCFs for liposome preparation is an
advanced and effective method, some disadvantages still need to
be overcome, such as the use of organic solvents (DELOS, SuperLip),
formulation of large liposomes (SCRPE, ISCRPE), wide particle size
distribution (SCRPE), and multistep preparation (SAS) [174].
Additional research in process design and liposome preparation
using supercritical fluids assisted methods on an industrial scale
is necessary, because of the low level of continuous production,
high investment costs, and disadvantages associated with the use of
SCFs. However, the influence of each parameter of the supercritical
fluids assisted process must be better investigated before liposome
production is carried out on an industrial scale [196].
Combined methods (supercritical assisted and conventional TE-
SC CO2 ) for liposome preparation are recommended.
Author Contributions
ML and MP wrote the manuscript, participated in conceptual-
ization and review & editing of the manuscript. ML, ŽK and MP
performed a literature review. All authors have read and approved
the final manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgments
The authors acknowledge the financial support of the Slovenian
Research Agency (research core funding No. P2-0046 - “Separation
Processes and Product Design” and research core funding No. J2-
1725 “Smart materials for bioapplications”).
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