International Journal of Pharmaceutics 601 (2021) 120571

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International Journal of Pharmaceutics

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Review

Design of liposomes as drug delivery system for therapeutic applications

Diana Guimarães a, Artur Cavaco-Paulo a, b, Eugénia Nogueira a, b, *
a
Centre of Biological Engineering, University of Minho, Braga, Portugal
b
Solfarcos – Pharmaceutical and Cosmetic Solutions, Braga, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: Liposomes are spherical vesicles consisting of one or more concentric phospholipid bilayers enclosing an aqueous
Liposomes core. Being both nontoxic and biodegradable, liposomes represent a powerful delivery system for several drugs.
Drug delivery They have improved the therapeutic efficacy of drugs through stabilizing compounds, overcoming obstacles to
Liposome production
cellular and tissue uptake and increasing drug biodistribution to target sites in vivo, while minimizing systemic
Liposome characterization
toxicity. This review offers an overview of liposomes, thought the exploration of their key fundamentals.
Market liposomes
Initially, the main design aspects to obtain a successful liposomal formulation were addressed, following the
techniques for liposome production and drug loading. Before application, liposomes required an extensive
characterization to assurance in vitro and in vivo performance. Thus, several properties to characterize liposomes
were explored, such as size, polydispersity index, zeta potential, shape, lamellarity, phase behavior, encapsu­
lation efficiency, and in vitro drug release. Topics related with liposomal functionalization and effective targeting
strategies were also addressed, as well as stability and some limitations of liposomes. Finally, this review intends
to explore the current market liposomes used as a drug delivery system in different therapeutic applications.

1. Introduction organized around an internal aqueous core, with the polar head groups
oriented to the inner and outer aqueous phase (Nisini et al., 2018). This
Liposomes were initial discovered in the 1960 s by the British he­ organized structure offers to liposomes the unique ability to load and
matologist Dr. Alec D. Bangham and collaborators at the Babraham deliver molecules with different solubility. Hydrophilic molecules in the
Institute, University of Cambridge, and the first report published in 1964 internal aqueous core, hydrophobic molecules into the lipid bilayer and
(Bangham and Horne, 1964). Liposomes are defined as a colloidal amphiphilic molecules at the water/lipid bilayer interface (Fig. 1)
spherical structure formed by self-assembly of amphiphilic lipid mole­ (Laouini et al., 2012a).
cules in solution, such as phospholipids (Sebaaly et al., 2016). Liposomal To date, liposomes have been investigated in several pharmaceutical
membrane can be composed of one or more lipid bilayers (lamellas) research as drug delivery systems and continue to constitute an intense

Abbreviations: AFM, Atomic force microscopy; AIDS, Acquired immunodeficiency syndrome; ASES, Aerosol solvent extraction systems; CH, Cholesterol; CO2,
Carbon dioxide; Cryo-TEM, Cryogenic – TEM; DLS, Dynamic light scattering; DOPC, 1,2-dioleoyl-snglycero-3-phosphocholine; DOPE, 1,2-Dioleoyl-sn-glycero-3-
phosphoethanolamine; DPPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol; DSC, Differential scanning calo­
rimetry; DSPC, 1,2-Distearoyl-sn-glycero-3-phosphocholine; DSPE, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; DSPG, 1,2-Distearoyl-sn-glycero-3-phospho­
glycerol; EDTA, Ethylenediaminetetraacetic acid; EE, Encapsulation efficiency; EPR, Enhanced permeability and retention; ESR, Electron spin resonance; FDA, Food
and Drug Administration; FFF, Field-flow fractionation; FTIR, Fourier transform infrared spectroscopy; GAS, Gas anti-solvent; GC–MS, Gas chromatography–mass
spectrometry; HPLC, High-performance liquid chromatography; LC-MS, Liquid chromatography–mass spectrometry; LDE, Laser Doppler electrophoresis; LUV, Large
Unilamellar Vesicles; MCL, Multicompartment liposome; MLV, Multilamellar Vesicles; MPS, Mononuclear phagocytic system; MVV, Multi Vesicular Vesicles; NMR,
Nuclear magnetic resonance; NTA, Nanoparticle tracking analysis; PA, Phosphatidic acid; PC, Phosphatidylcholine; PCS, Photon correlation spectroscopy; PDI,
Polydispersity index; PE, Phosphatidylethanolamine; PEG, Polyethylene glycol; PG, Phosphatidylglycerol; PI, Phosphatidylinositol; PS, Phosphatidylserine; RES,
Reticuloendothelial system; RESS, Rapid expansion of supercritical solutions; SAS, Supercritical anti-solvent; SAXS, Small-angle X-ray scattering; SCF, Supercritical
fluid; SCRPE, Supercritical reverse-phase evaporation; SEC, Size exclusion chromatography; SUV, Small Unilamellar Vesicles; Tc, Transition temperature of phos­
pholipids; TEM, Transmission electron microscopy; TGA, Thermogravimetric analysis; UPLC, Ultra-performance liquid chromatography; UV–Vis, Ultraviolet–visible;
XRD, X-ray diffraction.
* Corresponding author at: Centre of Biological Engineering, University of Minho, Braga, Portugal.
E-mail address: enogueira@[Link] (E. Nogueira).

[Link]
Received 15 February 2021; Received in revised form 25 March 2021; Accepted 31 March 2021
Available online 2 April 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.
D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

Phospholipid bilayer

Hydrophilic head
Hydrophobic tail
Aqueous
core

Hydrophilic molecules
Hydrophobic molecules

Amphiphilic molecules

Fig. 1. Representation of the general structure of liposomes.

field of research (Bozzuto and Molinari, 2015). Liposomes are consid­ in two different forms: natural and synthetic. The most natural phos­
ered a powerful drug delivery systems due to their structural versatility pholipids used to produce liposomes are PC and PE, that are abundant
as well as their biocompatibility, biodegradability, non-toxic and non- phosphatides in plants and animals (Antimisiaris et al., 2007). The main
immunogenicity nature (Mathiyazhakan et al., 2018). The amphiphilic sources of natural phospholipids are egg yolk or soya bean. Synthetic
character of phospholipids in solution mimic natural cell membranes, phospholipids are produced from natural lipids. Modification in head
allowing excellent interactions between liposomes and mammalian cell groups, aliphatic chains and alcohols of natural phospholipids creates a
membranes promoting an efficient cellular uptake (He et al., 2019). variety of synthetic phospholipids, that have proved to be more stable.
Additional advantages of liposomes include their ability to carry large Some examples of phospholipids in the synthetic form are 1,2-Dis­
drug payloads, capacity for self-assembly and a wide range of physico­ tearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glyc­
chemical and biophysical properties that can be modified to control ero-3-phosphocholine (DPPC), 1,2-dioleoyl-snglycero-3-phospho
their biological characteristics (Sercombe et al., 2015). choline (DOPC), 1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG),
Liposomes as a drug delivery system have improved therapies for a 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-Dioleoyl-sn-
range of biomedical applications by stabilizing therapeutic compounds, glycero-3-phosphoethanolamine, (DOPE) and 1,2-Distearoyl-sn-glycero-
overcoming obstacles to cellular and tissue uptake, and improving bio- 3-phosphoethanolamine (DSPE) (Monteiro et al., 2014a).
distribution of compounds to target sites in vivo (Ding et al., 2006; In an aqueous environment, phospholipids have a strong ability to
Hua and Wu, 2013). The drug loaded into liposome is protected against form stable bilayers due to their amphipathic character. Therefore, li­
physiologically occurring events, such as enzymatic degradation, posomes are formed by hydrophilic interactions between polar head
chemical and immunologic inactivation and fast plasma clearance, groups, van der Waals forces between hydrocarbon chains (keep the
contributing to improve and extension of its action. Since the drug is long hydrocarbon tails together) and hydrogen bonds with water mol­
inside the liposome, there is a minimization of its exposure to healthy ecules. Hydrophobic chains are repelled by water molecules and spon­
tissue, reducing the undesirable side effects compared with the free drug taneously occurs the self-assembly of liposomes in a closed bilayer
form (Bozzuto and Molinari, 2015). (Frézard, 1999; Monteiro et al., 2014a). Liposomes can be a combination
of two or more phospholipids and consisted of single or multiple lipid
2. Design of liposomes bilayers. Depending on the head of the phospholipids, liposomes can
acquire positive, negative, or neutral charges (Lombardo et al., 2016).
A suitable liposomal formulation can be achieved by choosing an The final liposomal properties are influenced by the structure and
adequate liposome composition, functionalization and even a targeting characteristics of phospholipids. Liposomes can achieve different func­
strategy, as developed deeper in the following sections. The selection of tionality with variations in head groups, aliphatic chains and in the
phospholipids, head group and chain length, as well as the ratio of li­ saturation of fatty acids (Liu et al., 2019). The stability of liposomes can
posomes components are crucial features to determine safety, stability, be promoted using phospholipids with longer tails, and low degrees of
and efficiency of liposomes (Kapoor et al., 2017). Moreover, the ability tail unsaturation and ether linkages. Phospholipids with longer satu­
of liposomes as drug delivery system can be affected by the number and rated hydrocarbon chains have higher ability to interact each other and
rigidity of lipid bilayers, size, surface charge, lipid organization and to form rigidly ordered bilayer structures. Otherwise, phospholipids
surface modification (Euliss et al., 2006; Sebaaly et al., 2016). with shorter unsaturated hydrocarbon chains that form liposomes with
fluid and disordered bilayers (Kapoor et al., 2017; Rawicz et al., 2000).
In addition to phospholipids, there are more liposomal components
2.1. Liposome components and properties
that can enhance the stability of liposomes such as cholesterol (CH),
glycols including propylene glycol and polyethylene glycol (PEG) and
The main component of liposomes are glycerophospholipids, which
even polymers as chitosan. These components can have pronounced
are amphiphilic lipids composed of a glycerol molecule bound to a
effects on healthy tissues and cells, as well as activate or suppress the
phosphate group and to two fatty acid chains that may be saturated or
immune system (Inglut et al., 2020). The incorporation of CH to the lipid
unsaturated (Pinot et al., 2014). The phosphate group can be also
bilayer of liposomes can influence the bilayer fluidity and rigidity
bonded to another organic molecule (Beltrán-Gracia et al., 2019; Mon­
reducing their permeability and increased their in vitro and in vivo sta­
teiro et al., 2014a). According to this organic group, natural phospho­
bility. CH, as a hydrophobic molecule, induces a dense packing of
lipids are classified as phosphatidic acid (PA), phosphatidylcholine (PC),
phospholipids and inhibits the interactions in the lipid chains by inter­
phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphati­
calating between them, promoting the stabilization of the liposomes
dylglycerol (PG) and phosphatidylserine (PS) (Tsuji et al., 2019). Glyc­
membrane (Bozzuto and Molinari, 2015; Lee et al., 2005; Sharifi et al.,
erophospholipids that are responsible to form liposomes can be divided

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

2019). CH molecule accommodates itself among the phospholipids with liposomes called targeted liposomes. A more detailed description of the
its hydroxyl group close to the hydrophilic region, and its aromatic rings different types of liposomes will be reported below.
parallel to the fatty acid chains into the lipid bilayer (Beltrán-Gracia
et al., 2019). CH is crucial for the structural stability of liposomes, in 2.1.1. Phase transition temperature
their absence liposomes can interact with proteins (albumin, transferrin, Another important parameter that can affect the fluidity of the lipids
macroglobulin and high-density lipoproteins). These interactions within the bilayer is the transition temperature of phospholipids (Tc),
destabilize the structure of the liposomal membrane and consequently which refers to the temperature at which phospholipids transit from gel
decreases their performance as drug delivery system (Lu et al., 2013; to liquid crystalline phase (Zamani et al., 2018). The Tc depends on the
Maranhão et al., 2017; Yingchoncharoen et al., 2016). length of the fatty acid chains, the degree of saturation of the hydro­
Another way to achieve the liposomal structure modification is the carbon chains, the ionic strength of the suspension medium and the
incorporation of glycols. Phospholipid vesicles with propylene glycol nature of the polar head group (Hussain et al., 2017; Maurer et al.,
have been advocated as flexible lipid vesicles in order to obtain delivery 2001). Lipid bilayers composed of phospholipids with long and satu­
systems for enhanced the skin delivery of drugs (Elmoslemany et al., rated hydrocarbon chains should be rigid and less permeable, due the
2012; Manconi et al., 2009). The use of different PEGs on the surface of interactions between the chains are stronger, resulting in a higher Tc.
liposomes can be a good approach to prolong blood circulation half-life Thus, hydrophobic interactions are stronger when the saturated hy­
from few minutes (conventional liposomes) to several hours (stealth li­ drocarbon tails increase in length (Akbarzadeh et al., 2013). At a tem­
posomes, also called PEGylated liposomes) (Beltrán-Gracia et al., 2019). perature lower than Tc, the phospholipids are in gel phase and
Indeed, one of the major drawbacks of conventional liposomes are their presenting low fluidity and low permeability, individual molecules
rapid clearance from the bloodstream and end up in organs and tissues within the bilayer move gently. At a temperature higher than Tc, the
in the reticuloendothelial system (RES) such as liver and spleen (Lee and phospholipids are in a liquid crystalline phase and having a high fluidity
Thompson, 2017). The increase in circulation lifetime of the liposomes and usually relatively low permeability, individual molecules within the
promoted by PEG has been found to depend on both the amount of lipid bilayer move quickly. At a temperature around Tc, the liposome
grafted PEG and the molecular weight or length of the polymer (Allen bilayer increases significantly the permeability due the presence of
et al., 1991). Usually, the longer-chain PEGs have produced the greatest highly permeable interfacial regions between coexisting gel and liquid
improvements in blood circulation time (Immordino et al., 2006). It was crystalline phase domains (Beltrán-Gracia et al., 2019; Collier and
reported that blood levels were higher for PEGylated liposomes with Messersmith, 2001).
longer molecular weight (PEG 1900 and PEG 5000) compared to
PEGylated liposomes containing a shorter chain (PEG 750 and PEG 120)
(Allen et al., 1991). The conformation of the PEG polymers on the sur­ 2.2. Liposome structure
face of the liposomes is determined by the PEG molecular weight and the
PEG surface density, and can be mushroom (low concentration) or brush According to their structure, liposomes are classified centered on the
(high concentration) regime (Perry et al., 2012). The increased in PEG number of lipid bilayers (lamellae) and on the vesicle size (Fig. 2). Based
concentration from 5% to 10% (molar ratio) showed a clearly improved on their lamellarity, liposomes can be classified as unilamellar (ULV, all
in the stealth degree of the liposomes. Liposomes that have a higher size range), multilamellar (MLV, >500 nm) and multivesicular (MVV,
concentration of PEG (brush regime) are more resistant to phagocytosis >1000 nm) vesicles (Akbarzadeh et al., 2013; Emami et al., 2016). ULV
and poorly activate the human complement system (Nogueira et al., can also be divided by their size into three categories, small unilamellar
2013). vesicles (SUVs, 20 – 100 nm), large unilamellar vesicles (LUVs, >100
The surface properties of liposomes improved by PEG are associated nm) and giant unilamellar vesicles (GUVs, >1000 nm). ULVs are char­
to a camouflaged effect, mimicking water-like structures, providing a acterized by the presence of a single bilayer, with more ability for the
steric barrier that prevents the adsorption of proteins in liposome sur­ encapsulation of hydrophilic compounds. MLVs present two or more
face and avoiding their recognition by macrophages of the mononuclear concentric lipid bilayers organized by an onion like structure, favorably
phagocytic system (MPS) that otherwise leads to a rapid liposome for the encapsulation of lipophilic compounds. MVVs include several
clearance (Kapoor et al., 2017; Monteiro et al., 2014a). Polymers such as small non-concentric vesicles entrapped within a single lipid bilayer and
chitosan is also used for the modification of the liposomal surface, are ideally suited for the encapsulation of large volume of hydrophilic
leading to a protective shell on the liposome surface, mainly for the oral material (Emami et al., 2016; Maherani et al., 2011). In addition to the
delivery of drugs (Caddeo et al., 2017; Henriksen et al., 1994). Besides vesicle size, the number of lamellae also affect the amount of certain
the incorporation of the components described below on their compo­ compound to be encapsulated in liposomes (Akbarzadeh et al., 2013;
sition, liposomes can be functionalized with specific ligands to improved Olusanya et al., 2018). Alternatively, an innovative vesicle-type
their ability as drug delivery system, leads to a new category of formulation is the multicompartment liposome (MCL). The MCL is
structurally composed of two different types of vesicles connected

Fig. 2. Liposomal classification based on lamellarity and size. SUV (Small Unilamellar Vesicles), LUV (Large Unilamellar Vesicles), MLV (Multilamellar Vesicles) and
MVV (Multi Vesicular Vesicles).

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

through a tight bilayer interface and are developed as single-vehicle degradation or oxidative alterations (Justo and Moraes, 2010). Ethanol
delivery systems for combinatory compounds (Al-Jamal and Kostar­ has also the additional benefit to be an acceptable solvent for in vivo drug
elos, 2007; Catalan-Latorre et al., 2016). delivery applications, at lower concentration, according to the European
pharmacopoeia (Marasini et al., 2017). Despite all the benefits, poor
3. Methods for liposome production and drug loading solubility of some lipids in ethanol, heterogeneity of liposomes when the
agitation fail, very low EE of hydrophilic compounds and incomplete
There are a great variety of techniques for liposome production, removal of ethanol from the liposomes, are the most concerns about this
including the liposomal formulation methods itself and the size reduc­ method (Çağdaş et al., 2014; Maherani et al., 2011). Numerous pa­
tion methods. The different techniques can influence the final properties rameters can be altered to control the particle size and EE obtained by
of liposomes, such as size, lamellarity, and encapsulation efficiency (EE) ethanol injection method, such as lipid nature, lipid concentration in
(Pattni et al., 2015). The methods to produce liposomal formulations can ethanol, drug to lipid ratio, diameter of injection orifice and injection
be categorized as conventional or novel. In the following section will be rate (Maherani et al., 2011; Wagner and Vorauer-Uhl, 2011).
explored some of these methods.
3.1.4. Detergent removal
3.1. Conventional methods The detergent removal method is another known technique to pro­
duce liposomes. In this method, phospholipids are solubilized with de­
Despite the vast gamma of conventional methods applied in liposome tergents at critical micelle concentrations (Isalomboto Nkanga et al.,
preparation, the most common used are the thin film hydration, reverse 2019). Upon detergent removal, by column chromatography or dialysis
phase evaporation, solvent injection, and detergent removal method bags, and with an adequate aqueous medium, phospholipids molecules
(Karn et al., 2013; Meure et al., 2008). These methods involve the self-assemble into liposomes (Akbarzadeh et al., 2013; Pattni et al.,
following basic stages: (i) lipids dissolved in organic solvents, (ii) 2015). Numerous parameters can influence the size and homogeneity of
removal of organic solvent, (iii) purifying and isolation of liposomes and the liposomes produced by this method, including initial ratio of phos­
(iv) analysis of final liposomes (Akbarzadeh et al., 2013). pholipids to detergents and rate of detergent elimination (Maherani
et al., 2011; Wagner and Vorauer-Uhl, 2011). The drawbacks of deter­
3.1.1. Thin film hydration gent removal method can be the presence of impurities in the final
The thin film hydration method, also known as the Bangham liposomal formulation, possible interaction between the detergent and
method, was the first described production process used in liposome the encapsulated compound and the fact of this technique to be very
technology (Bangham et al., 1967). In this simple method, lipids are time-consuming (Meure et al., 2008; Schubert, 2003).
initially dissolved in an organic solvent, generally chloroform, ether or
methanol, and dried down to form a thin lipid film in a round-bottom 3.2. Size reduction techniques
flask by organic solvent evaporation. The obtained thin lipid film is
hydrated using aqueous solvent and the liposomes are formed. Liposomes produced by most of the previous methods requires
Depending on hydration conditions, this method can create liposomes additional techniques to reduce their size, such as sonication, homoge­
with different structural organization. A vigorous shaking at hydration nization or extrusion (Kraft et al., 2014). There are two different soni­
process form MLVs with heterogeneous size, while a gentle hydration of cation techniques that can be used to control the size of liposomes, a
the lipid film generates GUVs (Isalomboto Nkanga et al., 2019; Monteiro bath and a probe sonication (Akbarzadeh et al., 2013). The sonication
et al., 2014b). The main drawbacks of this method are the production of process may have disadvantages as the difficult to provide identical
larger and heterogeneous liposomes, low entrapment ability, difficult to ultrasonic energy in a large volume of liposomal suspension (scale-up)
complete removal of organic solvent and to scale-up (Meure et al., and potential metal contamination from the probe tip. Furthermore,
2008). there is possible risk of degradation on phospholipids and even on
compound to be encapsulated, as well as low EE (Batzri and Korn, 1973;
3.1.2. Reverse phase evaporation Tejera-Garcia et al., 2011). In homogenization techniques, liposomes
One alternative method to prepare liposomes is the reverse phase can be forced to pass within an orifice through under high pressure to
evaporation. The initial procedure is the same of thin film hydration. reduce their size, resulting in a concept of high-velocity collision.
Phospholipids are dissolved in an organic solvent to form a film and then Several techniques can be included in this category of size reduction,
the solvent are removed by evaporation. The film is re-dissolved in an such as microfluidization, high-pressure homogenization, and shear
organic solvent (typically, diethyl ether and/or isopropyl ether), fol­ force-induced homogenization processes (Wagner and Vorauer-Uhl,
lowed by the addition of an aqueous phase, resulting in the formation of 2011). Another technique of reducing the size of liposomes is the
an oil-in-water emulsion (Pattni et al., 2015). The mixture is sonicated to extrusion process. After their formation, the liposomes pass several
produce inverted micelles, forming a homogeneous emulsion. The final times (extrusion cycles) through a membrane of defined pore size,
evaporation of the organic solvent under reduced pressure form a normally a polycarbonate filter, to uniform size distribution (Meure
viscous gel, that results subsequently into a liposomal suspension et al., 2008; Olson et al., 1979). This process requires much lower
(Akbarzadeh et al., 2013; Maherani et al., 2011). The advantage of this pressure and less volume of liposomal suspension compared with ho­
method is that permits a high EE (Monteiro et al., 2014b; Wagner and mogenizers (Kraft et al., 2014).
Vorauer-Uhl, 2011). The disadvantages include the exposure of the
compounds to be encapsulated to sonication conditions and even the 3.3. Novel methods
organic solvents (Antimisiaris et al., 2007). This method is also
described as time-consuming (Meure et al., 2008). The novel methods of liposome preparation are being investigated
mainly to facilitate the scale-up for industrial production and to be
3.1.3. Solvent injection techniques applied to a wide range of phospholipids and drugs (Pattni et al., 2015).
Liposomes can also be prepared by the solvent injection technique. There are novel methods based on the modification or improvement of
This method involves the quick injection of the lipids, dissolved in an conventional methods, such as cross-flow injection (Wagner) method
organic solvent (ethanol or ether), into an aqueous medium, resulting in (Wagner et al., 2002; Wagner and Vorauer-Uhl, 2011) and membrane
liposomes formation (William et al., 2020). The ethanol injection contractor technology, both modified/improved of ethanol injection
method is usual in liposomes production due its simplicity, reproduc­ method (Charcosset et al., 2005; Patil and Jadhav, 2014). The improved
ibility, fast implementation, easy scale-up and not cause lipid of detergent removal technique designs the cross-flow filtration method

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

Table 1 methods referenced above have an extremely potential future in the

Main characteristics of the novel methods for liposome production. therapeutic and pharmacological applications (Karn and Hwang, 2015).
Method Main characteristics The main characteristics, (+) advantages and (–) disadvantages of the
novel methods are outline in Table 1, based in literature reviewing (Has
Cross-flow injection (+) Simple, scalable, continuous and sterile process.
(Wagner) (–) Residual organic solvents can creates stability and Sunthar, 2019; Huang et al., 2014; Karn et al., 2013; Maherani et al.,
problems. 2011; Pattni et al., 2015).
(+) Simple, rapid, scalable and continuous process;
Membrane contractor homogenous liposomes with higher EE for lipophilic
3.4. Drug loading methods
drugs.
(–) Less studied for hydrophilic drugs; high-cost
material. As mentioned before, liposomes are considered a good drug delivery
(+) Rapid, scalable, sterile process; homogeneous system due their ability to load drugs with different characteristics
Cross-flow filtration liposomes with high stability; facility to removal of (Laouini et al., 2012a). The selection of an adequate method for drug
detergent.
encapsulation into liposomes depends of several factors such as EE,
(–) Understudy method.
(+) Sterile process; homogeneous liposomes by drug/lipid ratio, drug leakage and retention, sterility, facility of pro­
changing the nozzle diameter; narrow liposome size duction and scale-up, cost efficiency and liposome stability (Maherani
Injection and distribution; small organic solvent consumption. et al., 2011; Mayer et al., 1986). Furthermore, the amount of encapsu­
decompression (–) Complex equipment with low yield; needs of high
lated drug is related with the kind of drug, the composition of liposomes
temperature and pressure; more adequate for
hydrophobic drugs; nozzle can stay clogged.
and the method used for the liposomal production (Pattni et al., 2015).
(+) Simple fast and solvent-free process; liposomes with There are two different processes to encapsulate drugs into liposomes,
RESS controllable size. namely passive and active methods (Akbarzadeh et al., 2013).
(–) Low yield and EE. Passive loading method describes the procedure in which the drug is
(+) Suitable for a wide range of drugs; liposomes with
encapsulated during the liposome preparation. Hydrophilic drugs are
variable size; and moderate stability; solvent-free and
GAS uncontaminated process. dispersed in the aqueous phase (inside and outside of the liposomes),
(–) Require organic solvent and needs gas and solvent whereas hydrophobic drugs are located in the bilayer of the liposome
separation; batch process. (Maherani et al., 2011). In this procedure, immediately when they are
(+) Simple, scalable; solvent-free and uncontaminated being formed, liposomes can capture the aqueous volume containing the
process; homogenous, small and stable liposomes; low
SAS use of organic solvent and moderate pressure and
hydrophilic drug previous dissolved. Consequently, the concentration of
temperature. the drug inside the aqueous core is similar to the aqueous volume
(–) Require organic solvents and needs gas and solvent enclosed by the liposomes. The EE of drugs encapsulated by passive
separation; difficult to optimize conditions. loading changes due to numerous features, such as drug solubility,
(+) Rapid, scalable and single step process; more
liposome size and charge, lipid concentration and production method
ASES adequate for dry liposomes; low organic residues.
(–) Heterogeneous and large liposomes; uses a nozzle; (Pauli et al., 2019). The liposomal membrane is not permeable to ions
understudy method. and charged drugs. Otherwise, the uncharged drugs can diffuse through
(+) Simple, rapid and one-step process with scalable the lipid membrane, occurring drug leakage. Usually, this approach
SCRPE potential; no need for nozzles; reduced or no use of results in low EE, involving a large amount of non-encapsulated drug
solvent; stable liposomes.
(–) Understudy method; require high pressure; high-
and high drug leakage for the drugs permeable to liposomal bilayer (Li
cost material. et al., 2018). However, hydrophilic drugs that have protonizable amine
(+) Simple, rapid and reproducible process; functions can be encapsulated into liposomes by active loading,
Dual asymmetric homogeneous and small liposomes; high EE for water improving their EE in comparison with passive loading (Akbarzadeh
centrifugation soluble drugs.
et al., 2013).
(–) Used only for small volumes; only laboratory-scale,
not adequate for scale-up production, high pressure The principle of active loading, also called remote loading, involves
with agitation; understudy method. the creation of a transmembrane pH or ion gradient, that efficiently
(+) Scalable process and used for biological samples; drives the drug through the lipid bilayer, leading to up to 100% loading
liposomes with controllable size. in some drugs. This method is applied after the liposome formation. The
Microfluidics (–) Issues for thermolabile compounds; complex
equipment; not adequate for scale-up production;
gradient is created between the inside of the intact liposomes (already
difficult to clean after liposome production. formed) and the outside of liposome, the aqueous medium, where the
drug is solubilized. As uncharged drugs can diffuse across the lipid
membrane, they become protonated, inhibiting their diffusion out of the
(Karn and Hwang, 2015; Peschka et al., 1998). The direct hydration of liposome, enhancing their EE and retention inside liposome (Li et al.,
lipid components following the sonication process also represent an easy 2018). The ideal loading efficiency is achieved when the drug is an
method avoiding the use of dissipative steps (Manca et al., 2013; Man­ amphipathic weak base (pKa ≤ 11) or weak acid (pKa > 3) (Zucker et al.,
coni et al., 2003; Manconia et al., 2009). Furthermore, the use of su­ 2009). There are several approaches to performed active loading, such
percritical fluid (SCF) methods has been explored in liposomes as ammonium sulfate transmembrane gradient for amphipathic weak
production. These methods use a supercritical fluid, such as carbon di­ bases, calcium acetate gradient for weakly acidic drugs, phosphate
oxide (CO2), maintained under supercritical conditions (temperature gradient method, ethylenediaminetetraacetic acid (EDTA) gradient
and pressure). The SCF methods offers several advantages including a method and ionophore loading method (Li et al., 2018).
cheap and environmental harmless solvent, controlling of particle size,
in situ sterilization and possibility of large-scale production (Isalomboto 4. Characterization of liposomes
Nkanga et al., 2019; William et al., 2020). The most used SCF methods
are injection and decompression, rapid expansion of supercritical solu­ After production and before application, liposomes need to be
tions (RESS), processes with supercritical CO2 as an anti-solvent, gas extensively characterized for evaluation of their physico and chemical
anti-solvent (GAS), supercritical anti-solvent (SAS), aerosol solvent properties to guarantee their in vitro and in vivo performance (Çağdaş
extraction systems (ASES) and supercritical reverse-phase evaporation et al., 2014). The most investigated properties to characterize liposomes
(SCRPE) (Karn et al., 2013). Recently, other methods can be also are size, size distribution (reported using the polydispersity index, PDI),
employed to produce liposomes, such as dual asymmetric centrifugation surface charge (through zeta potential measurement), shape, lamellar­
and microfluidics (Huang et al., 2014; Meure et al., 2008). All the novel ity, phase behavior, EE, and in vitro drug release (Isalomboto Nkanga

5
D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

Table 2 determine the diffusion coefficient of particles based on the reads of the
Analytical techniques used for the evaluation of liposomal properties. intensity change of scattered light. Whereas, NTA find the diffusion
Properties Analytical techniques coefficient by the movements of individual particles in successive optical
video image. NTA can be a good approach to verify the size determined
Dynamic light scattering (DLS), Nanoparticle tracking
analysis (NTA), Nuclear magnetic resonance (NMR), Field- by DLS due they measure the same physical property. Therefore, the size
Size flow fractionation (FFF), Size exclusion chromatography measured by NTA should be similar to that observed in DLS technique
(SEC). Microscopy techniques: Transmission electron (Filipe et al., 2010; Malloy and Carr, 2006). The capacity of NTA to
microscopy (TEM), Cryogenic-TEM (Cryo-TEM) and simultaneously measure size and particle scattering intensity, besides
Atomic force microscopy (AFM).
Zeta potential Laser Doppler electrophoresis (LDE) and Capillary
allowing to distinguish particles of different refractive index within the
electrophoresis. same sample solution, makes a direct estimation of particle concentra­
Shape Microscopy techniques: TEM, Cryo-TEM and AFM. tion (Elizondo et al., 2011).
Cryo-TEM, 31P NMR, Small-angle X-ray scattering (SAXS)
Lamellarity and trapped volume determination techniques.
4.2. Zeta potential
Differential scanning calorimetry (DSC),
Phase behavior Thermogravimetric analysis (TGA), fluorescence probe
polarization, NMR, Electron paramagnetic resonance, The overall net charge of the particles is usually expressed as surface
Fourier transform infrared spectroscopy (FTIR) and X-ray or zeta potential (ζ-potential) (Kraft et al., 2014). This feature of lipo­
diffraction (XRD). somes is considered an essential physical property in the control of the
Ultraviolet–visible (UV–Vis) and Fluorescence
Encapsulation spectroscopy, enzyme or Protein-based assays, High-
electrostatic interactions between the particles in suspension (Kaszuba
Efficiency performance liquid chromatography (HPLC), Ultra- et al., 2010). The net charge of liposomes is influenced by key param­
performance liquid chromatography (UPLC), Liquid eters, such as lipid composition, the head group of lipids and associated
chromatography–mass spectrometry (LC–MS), Gas ligands, differing from negative, neutral, or positive. The ζ-potential can
chromatography–mass spectrometry (GC–MS), Electron
be also affected by the external environmental and its ionic strength
spin resonance (ESR) and 1H NMR.
Drug release Spectrophotometry methods, HPLC and UPLC. (Manconi et al., 2003). The ζ-potential measurements are used to predict
the stability of colloidal systems, such as liposomes in their surrounding
medium. Usually, liposomes with low ζ-potential or uncharged have
et al., 2019; Pattni et al., 2015). Table 2 summarizes the main analytical more probability to aggregate over time, because there will be no force
techniques used for the assessment of liposomal characteristics. to inhibit the liposomes flocculating. Otherwise, the liposomes in sus­
pension with a large negative or positive ζ-potential charge present
4.1. Size and polydispersity index repulsive forces in the medium that prevents the natural tendency to
aggregation (Laouini et al., 2012b).
The size and PDI of liposomes are the most relevant features in The measure of ζ-potential needs a laser to provide a light source to
liposome characterization. It has known that the liposome size shown to illuminate the liposomes within the sample. The laser beam passes
be a crucial factor for inhalation and parental administrations (Laouini through the middle of the sample cell used to the measurements at a
et al., 2012b) and to determine the circulation half-life of liposomes specific angle (Laouini et al., 2012b). Determination of the surface
(Elsana et al., 2019). While small liposomes can circulate in the organ­ charge permits the evaluation of fluctuations in the scattered light in­
ism for long time, large liposomes are more quickly eliminated from the tensity caused by the particle motion in the suspension due to the
blood circulation (Sercombe et al., 2015). For drug delivery, the desir­ application of an electric field. The charge of liposomes is proportional
able size of liposomes usually ranges between 50 and 200 nm (William to their mobility rate (Hadinoto et al., 2013; Pattni et al., 2015). Pos­
et al., 2020). The PDI value reveals in terms of size, the degree of sample teriorly, the information is passed to a digital signal processor in a
heterogeneity, that can be monodisperse or polydisperse. PDI can be computer system and the value of ζ-potential is calculated by deter­
dimensionless and scaled such that values range from 0 to 1. In drug mining the electrophoretic mobility, i.e. a velocity of a particle in an
delivery applications using liposomes, a PDI value equal or below 0.3 electric field, and then applying one specific equation, called Henry
indicates an acceptable and homogenous liposomal population (Danaei equation (Laouini et al., 2012b; Prabhu and Murugan, 2015). LDE and
et al., 2018), whereas high PDI value is associated with a very broad size capillary electrophoresis are the most known techniques used to mea­
distribution (heterogeneity) or even several liposomal populations in the sure the ζ-potential of liposomes through determination of their elec­
sample (Gaumet et al., 2008). The calculation of PDI is based on the trophoretic mobility (Monteiro et al., 2014b; Xiong et al., 2012).
particle size, refractive index of the solvent, the measurement angle and
the variance of the distribution (Koppel, 1972). 4.3. Shape
The most used technique to measure these two features is DLS also
known as photon correlation spectroscopy (PCS). DLS analyses the The analysis of morphological characteristics, namely the shape, is
continuous motion of the dispersed particles in solution (Brownian vital for an adequate characterization of liposomes. The most select tool
motion), resulting in scattering of the incident light. The scattering of to ascertain the morphological features of liposomes is the microscopy
the light is correlated with the diffusion level of the liposomes in sus­ (Elizondo et al., 2011). The visualization of liposomes as individual
pension (small particles diffused faster than the large particles). The particles by microscopy techniques provides a direct observation of their
evaluation of mean size is calculated based on the amount of light shape. Electron microscopy techniques such as TEM and cryo–TEM have
scattered. DLS is considered a simple, easy, fast and reliable method with been widely implemented for creating liposomal images (Isalomboto
the capacity to evaluate the liposome size in their native environment. Nkanga et al., 2019). TEM technique has some limitations at sample
Extensive range of measurement ability from a few nanometers to preparation level due the need to remove the native environment of li­
several micrometers is also applied (Isalomboto Nkanga et al., 2019; posomes. It is a time-consuming technique, thus is not flexible to being
Pattni et al., 2015). However, this technique has some limitations, routine measurements. Moreover, this technique may induce alterations
involving the difficult to differentiate single particles from aggregates in liposomal shape, including possible vesicle shrinkage, swelling or
and the high sensitivity to detect low amount of impurities (contami­ artifacts formation in the created image (Chetanachan et al., 2008;
nants) (Fissan et al., 2014). Pattni et al., 2015). To overcome these limitations, another possibility is
Recently, a size characterization tool called nanoparticle tracking the use of cryo–TEM. This approach keeps the liposomes close to their
analysis (NTA) was introduced to determine the size by measurement of native state and minimize the shape distortion or shrinkage by involving
the diffusion coefficient of particles in a sample (Kim et al., 2019). DLS the use of a flash freezing step with liquid nitrogen and then direct

6
D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

visualization of liposomes in a controlled environment. However, cry­ result of liposome preparation contains a mixture of encapsulated and
o–TEM usually works better with samples that have a lower nanometer non-encapsulated drug fractions. Thus, the first step to quantify the
range, because larger particles may be eliminated from the sample in the amount of drug within liposomes and consequently determined the EE is
preparation step. The AFM technique appears for direct analysis of li­ the separation of the free drug (non-encapsulated). Numerous tech­
posomes in their native environments without sample manipulation. It is niques have been used for this purpose, including size exclusion chro­
considered a quick, powerful and non-invasive technique (Laouini et al., matography based on the differences in size (liposome versus free drug),
2012b). The main advantage of this technique over electron microscopy gravitation or centrifugation, dialysis membrane with an appropriate
is the high resolution of the micrographs at three-dimensional level with cut-off and ultracentrifugation (Laouini et al., 2012b). The next step is
resolution down to the nanometer and Angstrom scales (Spyratou et al., the measurement of the amount of drug encapsulated into liposomes.
2009). There are two known ways to determine EE, namely the indirect and
direct method. The indirect method focuses on assessing the non-
4.4. Lamellarity encapsulated drug concentration in the eluted and subtract this con­
centration from the total drug concentration used in liposomal prepa­
Lamellarity is also a characteristic that can have an impact on the ration. Otherwise, in the direct method the determination of EE can
further liposomal applications due their influence on the EE and drug occur by direct disruption of liposomes with organic solvent and then
release profile. Cryo–TEM is the most used method and provide useful the released material is quantified (Bakonyi et al., 2017). The conven­
information regarding liposome lamellarity such as their bilayer thick­ tional techniques used to estimate the concentration of drug encapsu­
ness and inter-bilayer distance (Maherani et al., 2011). Other methods to lated into liposomes depends mainly on their nature and include UV–Vis
access the lamellarity are based on the visible or fluorescence signal and fluorescence spectroscopy, enzyme or protein-based assays (Laouini
variations of lipids marker upon the addition of certain reagents (Isa­ et al., 2012b). Moreover, the determination of the amount of drug can be
lomboto Nkanga et al., 2019; Laouini et al., 2012b). 31P NMR approach obtained using more sophisticate equipment such as HPLC, UPLC, liquid
has also been used to estimate the value of liposome lamellarity, chromatography and gas chromatography mass spectrometry (LC-MS
particularly, the ratio of phospholipid amount in the outer to inner and GC–MS, respectively) (Edwards and Baeumner, 2006). Additional
layers. The addiction of paramagnetic ions (Mn2+, Co2+, and Pr3+) to the techniques such as ESR and 1H NMR has also been used to quantify the
NMR sample preparation quenches the 31P NMR signal of the phos­ amount of drug (Anzai et al., 1990; Zhang et al., 2004).
pholipids. The interactions of the ions with the bilayer alter the NMR
spectrum. Therefore, by comparison of both spectrum, before and after 4.7. In vitro drug release
the incorporation of paramagnetic ions, it is possible to estimate the
lamellarity (Fröhlich et al., 2001). SAXS and trapped volume determi­ The evaluation of the in vitro drug release profile can be performed
nation are other techniques that also be used to estimate the lamellarity using dialysis conditions. The selection of dialysis bag membrane should
of liposomes (Mayer et al., 1985; Pattni et al., 2015). be in accordance with the drug specifications. It must be freely perme­
able to the drug and should not occur drug adsorption (Laouini et al.,
4.5. Phase behavior 2012b). Liposomal sample is placed into the dialysis bag with specific
molecular weight cut off, hermetically tied. The tubing membrane sys­
As mentioned above, the Tc represents an important feature that can tem is put into a simulated physiological fluid means release medium,
affect the fluidity of the lipid bilayer. For drug delivery applications, usually a buffered saline at pH 7.4. The full system is kept at 37 ◦ C to
phase behavior is highly considered due the fact that the lipid bilayer mimic an in vivo environment, and under continuous stirring. At defined
permeability to entrapped hydrophilic drugs increases with lipid mem­ time points, an aliquot of sample is taken and analyzed by the conven­
brane fluidity (Craig et al., 1990). Several other liposomal properties tional methods used for drug quantification. The volume of samples
including fusion, aggregation, stability and protein binding are also needs to keep constant. Thus, an equal volume of fresh release medium
dependent on the phase behavior of a liposomal membrane (Maherani is placed again in the system (Isalomboto Nkanga et al., 2019; Pattni
et al., 2011). Usually, the most common method used for study and et al., 2015). The data are used to establish the release profile by plotting
determination of the Tc is the DSC. This thermal analysis technique is the cumulative release percentage against the select time points. As
based on the evaluation of differences in heat flow, between a sample extrapolation to in vivo performance of liposomes as drug delivery sys­
reference and a study sample. Both samples are subjected to a pro­ tem, the results obtained from the in vitro release study are widely
grammed heating, cooling or isothermal treatment using a meticulous considered in the development of liposomes for the controlled release of
control of the atmosphere, typically saturated with nitrogen gas (Isa­ drugs (Dash et al., 2010).
lomboto Nkanga et al., 2019). The Tc can be also measure by other
methodologies such as TGA, fluorescence probe polarization, electron 5. Classification of liposomes
paramagnetic resonance, NMR, FTIR and XRD (Pentak, 2014; Sot et al.,
2005). To calculate the phase behavior of phospholipids in lipid bilayers Liposomes compared to others colloidal delivery systems offers the
can be also explored the molecular dynamics simulations (Youssefian advantage to alter their structural and physicochemical characteristics.
et al., 2017). Therefore, it is possible to modify liposomes behavior in vivo and tar­
geting liposomes to a specific site in the organism. Liposomes can be
4.6. Encapsulation efficiency classified based on their composition and functionalization. In addition
to conventional, stealth and targeted liposomes, the recent improvement
An optimal exploration of liposome characteristics may permit to on the design of liposomes leads to a different types of liposomes such as
develop liposomal formulations with ideal EE and allow the control of immunoliposomes and stimuli responsive liposomes (Allen and Cullis,
drug release. The liposome composition, the method of liposome pro­ 2013; Nisini et al., 2018). The differences between these categories of
duction as well as the rigidity of the bilayer membrane can have a liposomes will be highlighted below.
crucial impact on the EE of a certain drug (Maherani et al., 2011). Load
the proper amount of drug to achieve the therapeutic efficacy is the key 5.1. Liposome composition and functionalization
in the field of medical applications (Zucker et al., 2009). EE is calculated
as the percentage of the amount of drug inside liposomes (encapsulated Since their discovered, liposomes have been produced with different
drug), compared with the total amount of drug used in liposomal characteristics based on their composition and functionalization (Fig. 3).
preparation (encapsulated and non-encapsulated drug). The immediate The first generation of liposomes to be used in therapeutic applications

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

Conventional PEGylated
Hydrophilic
drug

Positively Polyethylene Glycol (PEG)

charged lipid
+
Negatively
charged lipid
-

Hydrophobic
drug

Antibody

Peptide
Small molecule

Protein
Targeting
ligand
Carbohydrate

PEG
Functionalized
Imaging agent

Ligand-targeted Multifunctional

Fig. 3. Different types of liposomes used in therapeutic applications.

was the conventional liposomes (Abra et al., 2002; Cattel et al., 2004; polymer onto the surface of the liposomes, the incorporation the PEG-
Immordino et al., 2006). These liposomes can be composed of neutral, lipid conjugate during liposome preparation, or thought the covalent
cationic or anionic charged phospholipids, usually in combination with attachment of reactive groups onto the surface of preformed liposomes
CH to promote the stabilization of the liposomal bilayer (Monteiro et al., (Immordino et al., 2006). However, an important restriction of stealth
2014a; Sercombe et al., 2015), as previous explained. However, this type liposomes is their large body biodistribution. Thus, the encapsulated
of liposomes continues to be subjected to several difficulties, such as the compound cannot be selectively delivered to specific target cells
instability in plasma which results in short blood circulation half-life. (Torchilin, 2005). From this limitation, ligand-targeted liposomes were
Liposomes are rapidly captured by RES and removed from the blood designed for targeted delivery of compounds at the desired tissues,
circulation (Immordino et al., 2006). The binding of opsonins, serum promoting higher and more selective therapeutic activity (Immordino
proteins, to the liposomes is the first signal for liposomes elimination. et al., 2006). In addition to surface modification of liposomes with PEG,
Opsonins, recognize the conventional liposomes as foreign particles, and targeted liposomes are also functionalized using glycoproteins, poly­
consequently they are destroyed by phagocytes of the MPS (Riaz et al., saccharides, or a ligand for specific receptors, such as antibodies, small
2018). molecules or peptides (Riaz et al., 2018; Torchilin, 2005). The ligand can
To overcome the difficulties of conventional liposomes, a second target specific receptors which are overexpressed on the surfaces of the
generation of liposomes was developed, led to the creation of so-called diseased cells, binding to them, resulting in a minimum off-target effects
stealth, long-circulating or PEGylated liposomes (Saraf et al., 2020). The to healthy cells (Fathi and Oyelere, 2016; Le et al., 2019).
stealth strategy involves mainly the possibility to coat the liposomal Following the principles of the previous strategy, it was considered
membrane surface with biocompatible hydrophilic polymer conjugates, the design of antibody-functionalized liposomes (immunoliposomes)
such as PEG, chitosan, and others, increasing repulsive forces between and stimuli-responsive liposomes (Nisini et al., 2018). Immunolipo­
liposomes and serum-components (Hatakeyama et al., 2013). Therefore, somes are formulated by chemically coupling of antibodies or their
results the reduction of immunogenicity and macrophage uptake, fragments to the liposomal surface, resulting in target antigens with an
enhancing its blood circulation half-life and reducing the toxicity of elevated degree of specificity (Eloy et al., 2017). In a stimuli sensitive
encapsulated compound (Madni et al., 2014). The methods to anchor the liposomal system, the release of the drug occurs upon changes in some
PEG in the liposome membrane involves the physical adsorbing of the physicochemical or biochemical stimuli, such as pH, temperature, redox

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

potential, enzymes and electrolyte concentrations, ultrasound, electric tissues or cells is performed by transport and delivery them into the
or magnetic fields (Drummond et al., 2000; Karanth and Murthy, 2007). tumor interstitium via leaky tumor vasculature through molecular drive
The most common examples of stimuli-responsive liposomes are the pH- within fluids (Gogoi et al., 2016). In this way, non-targeted liposomes
sensitive and temperature-sensitive liposomes (Li et al., 2010; Lu et al., ranging from 10 to 500 nm in size can accumulate preferentially on the
2014). Additionally to the delivery of drugs, liposomes can be used for tumor and inflamed tissues via the enhanced permeability and retention
other purposes, with simple modifications on their composition and (EPR) effect of the vasculature, because of abnormal leaky blood vessels
charge (Nisini et al., 2018). A good example is the use of cationic lipo­ and lack of functional lymphatics (Biswas and Torchilin, 2014; Fang
somes in gene therapy as transfection vectors, to the delivery of genes. et al., 2011; Torchilin, 2011). Passive targeting involves the needs to
The encapsulation of genes into liposomes, permits the protection of develop a liposomal formulation that can avoid their rapid elimination
nucleic acids against degradation during storage and in the systemic by organism defense mechanisms, such as phagocytic uptake or clear­
circulation (Immordino et al., 2006). ance by the cells of the MPS (Kraft et al., 2014). Thus, the preparation of
More recently, multifunctional liposomes have been studied for their stealth liposomes can be a good example to be used in passive targeting
potential to perform a combination of multiple functions through sur­ approaches due to surface modification of liposomes with PEG, that
face modification techniques, resulting in liposomes with a wide range permits increase their circulation time (Zylberberg and Matosevic,
of functionalities (Riaz et al., 2018). In literature have been reported 2016). This strategy, also involves the use of typical features of lipo­
several examples of multifunctional liposomes. One example is the somes, such as their charge, that can induce the specific targeting to the
theranostic liposomes, at the same liposome it is possible to have an cancer cells. Another example, can be the cationic liposomes. This type
imaging and therapeutic agent (diagnosis and treatment functions) (Li of liposomes is found to bind the negatively charged phospholipid head
et al., 2012; Sercombe et al., 2015). Another example is the dual- groups, specially expressed on tumor endothelial cells by electrostatic
targeting liposomes that involves liposomes having two different li­ interactions (Byrne et al., 2008). The mechanism of targeting based only
gands (Riaz et al., 2018). on the EPR effect is not enough to complete attenuate the side effects of
cytotoxic drugs. The heterogeneity of EPR effect within tumors and their
limitation to some solid tumors, can also affect the efficacy of drugs
5.2. Targeting strategies of liposomes
delivered by passive targeting (Kraft et al., 2014; Park et al., 2019).
Therefore, the development and searching of alternative targeting ap­
Almost as intense, an area of research and development of the lipo­
proaches with advanced functionalities such as active targeting have
some formulation is their targeting strategies. The specific targeting is a
been explored (Wicki et al., 2015).
primordial functional property of liposomes as drug delivery systems
(Zylberberg and Matosevic, 2016). Thus, targeting of specific sites fo­
5.2.2. Active targeting
cuses on both the development of new diagnostic tools and improving
In 1906, the visionary Paul Ehrlich introduced the concept of active
the efficacies of therapeutic agents (Fay and Scott, 2011). Currently,
targeting by describing a “magic bullet” needed to direct specific drug
there are two main strategies by which targeting of liposomes can be
delivery within the body (Lehner et al., 2013; Strebhardt and Ullrich,
broadly classified, namely passive and active targeting. Passive tissue
2008). Since then, researchers worldwide have been searching for the
targeting is mainly achieved through properties of cancer vasculature,
“magic bullet” that would target selected cells with precision facilitating
and active tissue targeting through receptor-specific ligands on the
diagnosis and therapy (Bazak et al., 2015).
liposome surface intended for cell binding (Fig. 4) (Lehner et al., 2013).
Active targeting involves the attachment of a targeting ligand to the
surface of liposomes for enhanced delivery of liposomal systems (Riaz
5.2.1. Passive targeting
et al., 2018). Numerous targeting ligands have been employed to active
Passive targeting approaches have been mainly applied in the
targeting, including antibodies, nucleic acids (aptamers), peptides and
oncology field due to pathophysiological features of cancers and their
whole proteins (e.g., transferrin) and small molecules such as vitamins
environment (Wicki et al., 2015). Passive targeting of liposomes to

Fig. 4. Schematic illustration of passive and active targeting strategies of liposomes into a tumor for enhancing the therapeutic efficacy of drugs.

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

(e.g., folic acid) (Wicki et al., 2015). There are several aspects considered reactions that can be stimulated by light, metal ions or temperature
in the selection of target ligands, which include: relative degree of over- (Monteiro et al., 2014a). Chemical degradation may induce perme­
expression or selective expression on the target, target cell uptake of the ability changes within lipid membrane. Additionally, the interactions
ligand-targeted formulation, and degree of covering of the target between the drugs and phospholipids can also interfere in liposomal
molecule (Noble et al., 2014; Sawant and Torchilin, 2012). These li­ chemical stability. The control of microbial stability of liposomal for­
gands should also be selected to allow binding to the target cells while mulations is also important due the therapeutic formulations of lipo­
minimizing binding to healthy cells (Lehner et al., 2013; Torchilin, somes are parenteral products and must be sterilized to remove the
2005). microbial contaminants from the final product (Laouini et al., 2012b).
There are three main approaches available to functionalize lipo­
somes. The first is binding the desired targeting ligand to a lipid prior to 6.1.1. Freeze-drying
mixing them with other lipid components during liposome preparation. Liposomal formulations can be stored in an aqueous solution or in a
In second approach, immediately after preparation, liposomes are dry powder form (Sebaaly et al., 2016). To overcome the main insta­
functionalized with the required targeting ligand (Marqués-Gallego and bility issues of liposomes in an aqueous solution, their storage in a dry
De Kroon, 2014). Head group modified lipids with a PEG spacer func­ state can be an attractive way for long-term stability (Chen et al., 2010).
tionalized at the end with amine, carboxylic acid, thiol or maleimide Among the feasible methodologies, freeze-drying, also known as
groups represent available options for this approach (Conde et al., lyophilization, remains the most studied and applied technique for this
2014). In another methodology, it was proposed the post-insertion of the purpose. Freeze-drying consists on water removal from a frozen sample
functionalized lipid in preformed liposomes. This method is based on the by sublimation and desorption under vacuum. However, the complexity
spontaneous incorporation of functionalized lipids from the micellar of the process itself can compromise the liposomal membrane integrity
phase into preformed and even drug-loaded liposomes. Derivatization of from stresses caused by the freezing and drying steps. Thus, the choice of
the targeting molecule happens in a separated step, as an approach to an ideal conditions to lyophilization is the main challenge to origin a
prevent the interference of activated lipids with other liposomal com­ final product with adequate characteristics, such as (i) elegant cake
ponents such as those present in the buffer (Steenpaß et al., 2006). appearance with fast reconstitution time, (ii) suitable physico-chemical
characteristics after reconstitution, (iii) low water content and (iv)
6. Limitations of liposomes satisfactory long-term stability of final liposomal formulation (Abdel­
wahed et al., 2006). The use of an appropriate excipient within the
The stability of liposomes is a key consideration in drug delivery liposomal formulation, can maintain their size and avoid their drug
applications. Indeed, the therapeutic effect and safety of liposomes leakage (Janicki et al., 2002). The excipients are included to protect the
encapsulating drugs depend on their lifetime and their distribution liposomes in the main steps of the freeze-drying process, cryoprotectants
within the body, and these features are directly related with their sta­ assist in freezing stress and lyoprotectants contribute in drying stress.
bility (Taira et al., 2004). The stability is considered the main concern Table 3 represents the most used excipients in freeze-drying of phar­
for liposome preparation, storage and further administrations steps maceutical products (Abdelwahed et al., 2006).
(Laouini et al., 2012b). The potential instability issues of liposomes are The formulation features, including the liposomal composition, the
typically related to oxidation and/or hydrolysis of lipids, drug leakage, nature of the drug as well as the type of excipient are the main
aggregates formation or even liposomal fusion (Immordino et al., 2006). responsible by the protective effect during lyophilization. Therefore, an
Another challenge of liposomal formulations is the identification of a exhaustive optimization of these features can be an appropriate way to
suitable large-scale production method and the needs to found an effi­ improve the stability of the liposomes after lyophilization. The most
cient sterilization technique for liposomes. These limitations of lipo­ used excipients are the sugars such as trehalose, sucrose and glucose.
somal formulations will be addressed below. The sugars are considered ideal stabilizers to protect liposomal integrity
during the lyophilization process. The stabilizer effect promoted by
6.1. Liposome stability sugars depends on their nature and concentration. Thus, these param­
eters must be careful selected and optimized to guarantee an enhanced
Liposomes itself are considered a moderately unstable colloidal stabilizer effect of lyophilized liposomes (Fig. 5) (Abdelwahed et al.,
system. A stable liposomal form preserves its physical integrity and does 2006).
not negatively stimulus the chemical integrity of the encapsulated drug
during its life (Laouini et al., 2012b). The evaluation of liposomal sta­
bility includes the verification of some specific parameters such as (i) the
chemical and physical stability, (ii) the conservation of their size and Table 3
structure, (iii) the maintenance of encapsulated drug and (iv) the impact Examples of commonly used excipients in freeze-drying of pharmaceutical
of biological fluids on the liposomal properties (Monteiro et al., 2014a). products.
Thus, these parameters can be interrelated. According to Food and Drug Type of Main characteristics Excipient
Administration (FDA), liposomes needs to be stable at least two years to excipient
be considered a liposomal drug product (Maherani et al., 2011). Bulking agents Offers bulk to the Trehalose, mannitol, lactose,
Chemical and physical stability are the main critical issues that in­ formulations, in the case of hydroxyethyl starch and
fluence the final performance of liposomes at biological level (Anti­ very low concentration of the glycine.
misiaris et al., 2007). Normally, the evaluation of size and the visual product to be freeze.
Regulate pH changes during Phosphate, tris hydrochloride,
inspection of liposomes appearance are two principal features to Buffers freezing. citrate and histidine.
determine the liposomal physical stability. This event is related to the Stabilizers Protect the liposomes during Sucrose, lactose, glucose,
tendency to agglomeration or aggregation. Thus, fusion and breakage of the lyophilization process, trehalose, glycerol, mannitol,
liposomes on storage can also leads to drug leakage from liposomes including freezing drying sorbitol, glycine, alanine,
stresses. lysine, PEG and dextran.
(Laouini et al., 2012b). Chemical stability can be considered the aptitude
Tonicity Control the osmotic pressure Sucrose, mannitol, glycine,
of liposomes to preserve the level of EE when changes in the medium can adjusters and produce an isotonic glycerol and sodium chloride.
be occur, including pH alterations, electrolyte composition, oxidizing solution
agents, and presence of surface active compounds (Maherani et al., Collapse Obtain higher drying Hydroxypropyl-
2011). The most important component in liposomes is the lipid. In its temperature temperatures increasing the β-cyclodextrin, PEG and
modifiers collapse temperature. dextran.
turn, lipids contain unsaturated fatty acids and can suffer oxidative

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

Without cryo/lyoprotection

Freeze-drying

(Dry state)

Homogeneous liposomes Heterogeneous liposomes

With cryo/lyoprotection

Freeze-drying

(Dry state)

Homogeneous liposomes Homogeneous liposomes

Fig. 5. Effect of cryo/lyoprotection on the size of liposomes after the freeze-drying process.

6.2. Scale-up and sterilization methods liposomal formulation and should be destructive for the microorganisms
(Barenholz and Lasic, 2018). The most common technique to achieve
The major limitation of liposomes application is the identification of sterilize small liposomes is the filtration using a sterile polycarbonate
a suitable method for large scale production as known as scale-up. To membrane with adequate pore size, normally 0.22 µm, under aseptic
use liposomes as an acceptable pharmaceutical product, their produc­ conditions (Laouini et al., 2012b). This method has the advantage that is
tion at large scale needs to be easily and economically feasible (Laouini not destructive for small liposomes. Filtration is not appropriated for
et al., 2012b). The slowed develop in scale-up process is associated to liposomes with high values of size (>0.22 µm) and for large volume of
the time dispensed to resolve problems involving the quality and tech­ liposomes due the possibility of filter clogging which compromises the
nological control. These problems included (i) accessibility of high- final product. It should be noted that there are other methods for lipo­
quality lipid raw materials, (ii) validated quality control analyses, (iii) somes sterilization, for example, autoclaving, high pressure sterilization
unavailability of equipment, (iv) reliability and reproducibility batch to using nitrogen gas, utilization of saturated steam to sterilize pharma­
batch, (v) efficient and valid sterilization methods and (vi) long-term ceutical equipment, ethylene oxide treatment, UV sterilization, γ-irra­
stability of produced liposomes. All these problems can be interrelated diation and dense gas technique (Barenholz and Lasic, 2018; Toh and
(Barenholz and Lasic, 2018; Saraf et al., 2020). As discussed in section 3, Chiu, 2013). However, it is important to note that conditions required in
there are several methods available for production of liposomes at lab­ these conventional sterilization techniques can be detrimental to the
oratory scale. However, only a few manufacturing techniques are stability of the liposomal preparations (Toh and Chiu, 2013).
available at industrial scale (Wagner and Vorauer-Uhl, 2011). The
production of liposomes involves an amount of unit operations which 7. Therapeutic applications of liposomes
are not easy to transpose for commercial manufacturing (Toh and Chiu,
2013). The key issue for production of a successful liposomal formula­ Liposomes have been revealing promising results as drug delivery
tion at industrial scale is the control and keep constant the character­ system for numerous kinds of drugs. Thus, the intensive investigation of
istics of each batch maintaining the reproducibility of the method liposomes in medicine led the researches to develop different liposomal
(Langer et al., 2008). At laboratory scale, usually is easy to reach the formulations for the controlling and management of a wide range of
reproducibility of the process, whereas at industrial scale the PDI of li­ diseases besides an extensive variety of therapeutic applications. The
posomes is difficult to control and the reproducibility of batch-to-batch encapsulation of drugs inside liposomes improve their therapeutic effect
is challenging to achieve (Zamboni et al., 2012). The ethanol injection due the pharmacokinetics and pharmacodynamics alterations (Bulbake
method is considered the most interesting technique for this purpose due et al., 2017). The modulation of the in vivo drug behavior and the
the reproducibility and fast implementation of this method (Charcosset reduction of the drug toxicity in the organism are the crucial features to
et al., 2015; Justo and Moraes, 2011). design a suitable liposomal formulation. The use of liposomes in clinical
Another limitation of liposomes is their sterilization that remains a applications focuses in the treatment and diagnosis of cancer. However,
challenging issue due the susceptibility of liposomes to physical and the potential of liposomes for therapeutic applications is not limited to
chemical degradation. Methods for liposomes sterilization should be a cancer therapy. Liposomes are considered an extremely flexible platform
compromise between the inactivation of the microorganism’s contami­ and can be used in diverse field of research (Maurer et al., 2001). In this
nation and the degradation of liposomal product. The sterilization section will be explored the current market liposomes and the use of
methods should not affect the physical and chemical characteristics of liposomes specifically in rheumatoid arthritis therapy.

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D. Guimarães et al. International Journal of Pharmaceutics 601 (2021) 120571

Fig. 6. Main therapeutic fields covered by liposomal formulations products (adapted from (Bulbake et al., 2017)).

7.1. Marketed liposomes for numerous kinds of drugs. The direct application of liposomes in
medicine encourages the researchers to create novel liposomes for
Liposomes have revealed significant therapeutic benefits in clinical treatments and diagnosis in a wide range of diseases as well as in a
applications. However, their applicability is limited due to the all stages variety of therapeutic applications. In the context of liposomal therapy,
of liposomal development and production process that comprises the modulation of the in vivo drug behavior and the reduction of the drug
manufacturing methods, regulatory approval by the competent author­ toxicity in the organism are the crucial features to design a proper
ities and intellectual property (Saraf et al., 2020). Despite all the liposomal formulation. A suitable liposomal formulation product con­
intensive research in the development of liposomal formulations to use sists in three essential components, lipids to form a liposome, molecules
in therapeutic application, in the moment, only a few liposomes have to functionalized them and a drug molecule that will be encapsulated. As
entered in the market as a commercialized liposomal product (Moo­ we can see from this review, the development and improvement of li­
savian et al., 2019). posomes are a complex challenge that involves the simultaneous opti­
The first successful liposomal formulation, Doxil®, was introduced to mization of several parameters to achieve a final liposomal formulation
the USA market in 1995 and it is the first liposomal product to obtain safe and effective. Although there are actually some liposomes approved
regulatory approval by FDA. Doxil®, or Caelyx® in Europe, is an on the market covering many health areas, it is possible to claim that
intravenous injection product that contain doxorubicin (DOX) hydro­ there is still much to be done in the field of liposomal technology to
chloride in their formulation. Doxil® is used to treat advanced ovarian overcome the limitations explored in this review. In summary, lipo­
cancer and acquired immunodeficiency syndrome (AIDS)-associated somes can contribute to treatments with key performance, hence it shall
Kaposi’s sarcoma, after the inefficiency of prior chemotherapy or lead to a better clinical outcome, lower toxicity levels and fewer side
intolerance therapies (Bulbake et al., 2017). These liposomes proved to effects.
improve the pharmacokinetic properties of free DOX and minimize the
life-threatening toxicities caused by the drug. Despite cancer therapy is CRediT authorship contribution statement
the most studied area in terms of liposomal clinically approved products,
liposomal products were also investigated for other diseases. Fig. 6 Diana Guimarães: Conceptualization, Investigation, Writing -
identifies the main therapeutic fields covered by liposomal formulations original draft. Artur Cavaco-Paulo: Supervision, Project administra­
products (Bulbake et al., 2017). The product name, active agent and tion, Funding acquisition. Eugénia Nogueira: Supervision, Conceptu­
pharmacological indications are also referenced. It can be prepared in alization, Writing - review & editing. : .
different forms, liquid (suspension), solid (dry power) and semi-solid
(gel or cream). The administration of liposomes in vivo can be topi­
Declaration of Competing Interest
cally or via parenteral route (Laouini et al., 2012b).
It is important to highlight that most of liposomal products devel­
The authors declare that they have no known competing financial
oped are nowadays under different pre-clinical studies and clinical tri­
interests or personal relationships that could have appeared to influence
als. The translation of liposomes for clinical trials requires advanced
the work reported in this paper.
models and methodologies. These models can predict the biosafety of
liposomes inside the organism to enhanced their therapeutic applica­
tions (Saraf et al., 2020). Acknowledgments

8. Conclusion This work has received funding from the European Union Horizon
2020 research and innovation programme under grant agreement NMP-
Liposomes have gained extensive attention as drug delivery system 06-2015-683356 FOLSMART. This study was also supported by the
Portuguese Foundation for Science and Technology (FCT) under the

12
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scope of the strategic funding of UIDB/04469/2020 unit and Bio­ Cattel, L., Ceruti, M., Dosio, F., 2004. From conventional to stealth liposomes: A new
frontier in cancer chemotherapy. J. Chemother. 94–97. [Link]
TecNorte operation (NORTE-01-0145-FEDER-000004) funded by the
[Link]-1.94.
European Regional Development Fund under the scope of Norte2020 - Charcosset, C., El-Harati, A., Fessi, H., 2005. Preparation of solid lipid nanoparticles
Programa Operacional Regional do Norte. Diana Guimarães (SFRH/BD/ using a membrane contactor. J. Control. Release 108, 112–120. [Link]
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Charcosset, C., Juban, A., Valour, J.P., Urbaniak, S., Fessi, H., 2015. Preparation of
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