Advanced Drug Delivery Reviews 176 (2021) 113851

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

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

Liposome composition in drug delivery design, synthesis,

characterization, and clinical application q
Danielle E. Large, Rudolf G. Abdelmessih, Elizabeth A. Fink, Debra T. Auguste ⇑
Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., Boston, MA 02115, USA

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

Article history: Liposomal drug delivery represents a highly adaptable therapeutic platform for treating a wide range of
Received 23 April 2021 diseases. Natural and synthetic lipids, as well as surfactants, are commonly utilized in the synthesis of
Revised 18 June 2021 liposomal drug delivery vehicles. The molecular diversity in the composition of liposomes enables drug
Accepted 22 June 2021
delivery with unique physiological functions, such as pH response, prolonged blood circulation, and
Available online 2 July 2021
reduced systemic toxicity. Herein, we discuss the impact of composition on liposome synthesis, function,
and clinical utility.
Keywords:
Ó 2021 Elsevier B.V. All rights reserved.
Liposomal drug delivery
Liposome synthesis
Liposome functionality

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Liposome synthesis & characterization methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Liposomes classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. Methods of liposomes preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2.1. Thin film hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2.2. Reverse-phase evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2.3. Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2.4. Detergent removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2.5. Dehydration-rehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2.6. pH jumping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2.7. Hydration in a packed bed of colloidal particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2.8. Freeze-thaw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3. Methods of liposome characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3.1. Drug encapsulation efficiency and release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3.2. Size, zeta potential, and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3.3. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. Molecular components of liposomal drug delivery vehicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1. Natural lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1.1. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1.2. Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.3. Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.4. Polysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2. Synthetic lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.3. Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Effect of composition on liposome characteristics & functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

q
This review is part of the Advanced Drug Delivery Reviews theme issue on
‘‘Transl Drug Delivery”.
⇑ Corresponding author.
E-mail address: d.auguste@northeastern.edu (D.T. Auguste).

https://doi.org/10.1016/j.addr.2021.113851
0169-409X/Ó 2021 Elsevier B.V. All rights reserved.
D.E. Large, R.G. Abdelmessih, E.A. Fink et al. Advanced Drug Delivery Reviews 176 (2021) 113851

4.1. Drug encapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4.2. Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.3. Surface charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.4. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.4.1. Clearance rate and half-life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.4.2. Biodistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5. Liposomal composition imparts unique functionalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.1. pH responsive liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2. Temperature responsive liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.3. Theranostic liposomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6. Liposomal drugs in clinical use and preclinical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6.1. Anti-cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6.2. Pain management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6.3. Anti-bacterial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6.4. Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction electroformation in microfluidics, pulsed microfluidic jetting, tran-

Since Alec D. Bangham’s discovery of liposomes in 1965, the
lipid vessel has become a widely utilized vehicle to encapsulate
and deliver molecules to treat a variety of diseases. The primary
component of liposomes are lipids and fatty acids that, due to their
natural occurrence in cell membranes, are considered inherently
biocompatible and biodegradable [1]. Structurally, liposomes are
defined by the self-assembly of amphipathic molecules into a
bilayer sphere, in which the hydrophilic head groups face the exte-
rior aqueous environment and the hydrocarbon chains assemble
within the hydrophobic interior. The amphiphilic character of lipo-
somes makes them ideal drug carriers for molecules of differing
polarities. Liposomal encapsulation of drugs reduces systemic tox-
icity and improves tolerable dose regimens for anti-cancer, anti-
bacterial, and anti-fungal therapies [2–4]. The lipid chemistry is
critical for optimizing drug encapsulation, stability, and release,
and liposome pharmacokinetics and pharmacodynamics. Herein,
we present a review of the literature focused on the rational design
of liposomes based on chemical, mechanical, and physiological
properties.
2. Liposome synthesis & characterization methodology
2.1. Liposomes classification
Two important characteristics of liposomal vesicles that influ-
ence drug encapsulation efficiency and circulation time are size
and membrane lamellarity [1,5–7]. The method of synthesis deter-
mines the type of liposomes produced. Liposomes are classified as
unilamellar vesicles (ULVs) with one bilayer membrane, oligo-
lamellar vesicles (OLVs) with 2–5 bilayer membranes, and multil-
amellar vesicles (MLVs) with five or more bilayer membranes.
ULVs are further categorized into small unilamellar vesicles (20–
100 nm in diameter, SUVs), large unilamellar vesicles (100 nm-
1 mm, LUVs), and giant unilamellar vesicles (>1 mm, GUVs). SUVs
exhibit uniform drug encapsulation and release kinetics along with
longer circulation times; therefore, they are the most commonly
used as drug delivery vehicles [8].
2.2. Methods of liposomes preparation
Liposome synthesis is a heavily investigated area of research
with many recent and modified techniques, including: heating,
curvature tuning, localized IR heating, osmotic shock, dual asym-
metric centrifugation, spray-drying, lyophilization, gel-assisted
hydration, hydration on glass beads, hydration in microfluidics,
2
sient membrane ejection, continuous droplet interface crossing
encapsulation and stationary phase interdiffusion method [6].
Herein, we discuss the most common methods for bench scale
preparation of liposomes [1,5,6].
2.2.1. Thin film hydration
The most common method employed for liposome synthesis is
thin film hydration [9–12]. In this method, lipids and amphiphilic
molecules are solubilized and mixed in an organic solvent. The
mixture is then transferred into a round-bottom flask and the sol-
vent is evaporated using a rotary evaporator under vacuum, leav-
ing a thin film of lipids. The thin film is then hydrated in a
solution that may contain one or more hydrophilic drugs that are
desired to be encapsulated. The temperature of the hydration buf-
fer must be above the gel-liquid phase transition temperature (Tm)
of the lipid. The volume of the aqueous solution used to hydrate
the lipid film affects the characteristics of the formed liposomes;
large volumes lead to the formation of MLVs while the rate of
hydration determines the efficiency of drug encapsulation [5].
The slower the rate of hydration the higher the encapsulation
efficiency.
The size and lamellarity of the vesicles may be controlled by
either extrusion through membranes of specific pore sizes [13] or
the use of sonicators, where the frequency of the ultrasonic waves
and the duration of the process determine the size distribution of
the fabricated liposomes [14]. A jacketed extruder or water bath
may be used to maintain the solution temperature above the Tm
of the lipid if necessary. Although sonication is easier and more
convenient for post-synthesis processing to produce SUVs, espe-
cially when large volumes are needed, it results in less uniform
liposomes with lower drug encapsulation efficiency compared to
those produced by extrusion [1]. Sonication may also degrade
lipids or drugs encapsulated, partly due to the heat generated dur-
ing the process. Furthermore, drug-loaded SUVs made by thin film
hydration followed by extrusion are often stable for longer periods
than their equivalents made by sonication or detergent removal
methods [1].
2.2.2. Reverse-phase evaporation
Reverse-phase evaporation produces a mixture of LUVs and
MLVs entrapping large aqueous volumes, which allows for encap-
sulation of large molecules, such as proteins and nucleic acids [1].
In this method, lipids and amphiphilic molecules are first mixed in
an organic solvent [15], then an aqueous buffer, which may contain
a solubilized drug, is added to the mixture. Afterwards, the organic
solvent is evaporated using a rotary evaporator under low
D.E. Large, R.G. Abdelmessih, E.A. Fink et al.
pressure, leaving the lipid vesicles dispersed in the aqueous solu-
tion. If an application requires smaller, more uniform particles,
the size of liposomes may be reduced by extrusion [16]. In this
case, the pore size of the polycarbonate filter and the number of
extrusion cycles will determine the size and polydispersity of the
synthesized liposomes [16,17].
2.2.3. Injection
There are several variations of the injection method [6]. In one
technique [18], lipids and amphiphilic molecules are dissolved in
an organic solvent of a low boiling point (e.g., diethyl ether) then
the mixture is injected into a warm aqueous solution whose tem-
perature is constant and above the boiling point of the solvent
used. This allows the organic solvent to evaporate and lipid vesicles
to form, producing primarily LUVs. Alternatively, if the organic sol-
vent used has a higher boiling point (e.g., ethanol), the solvent-
lipid mixture may be injected into an aqueous solution at room
temperature under constant stirring [19], and the organic solvent
can then be removed via dialysis [20] or filtering. This method is
most expedient for preparing large volumes of liposomal formula-
tions; however, the fabricated liposomes usually have higher poly-
dispersity indexes (PDI), suggesting a wide distribution of sizes,
and in many cases MLVs may be present [1].
2.2.4. Detergent removal
Several techniques of the detergent removal method may be
used to synthesize LUVs. In this method, lipids and amphiphilic
molecules are mixed with a surfactant characterized by a high crit-
ical micelle concentration (CMC) in an organic solvent which is
then evaporated under low pressure [5,21]. A solution of mixed
micelles is obtained by hydrating the lipid film in an aqueous buf-
fer. The detergent is then removed by means of dialysis [22–25],
size-exclusion chromatography [26–28], adsorption onto
hydrophobic beads [29] or dilution [30,31]. The sample is then con-
centrated which allows the lipids to form LUVs.
2.2.5. Dehydration-rehydration
Dehydration-rehydration is used to synthesize LUVs without
the use of organic solvents or detergents. In this method, liposomes
are made by dispersing lipids and amphihilic molecules at low con-
centrations directly into an aqueous solution followed by sonica-
tion [32]. The drug to be encapsulated may be added to the
aqueous solution and mixed with the formulated vesicles. When
water is evaporated under nitrogen, liposomes combine, creating
a multilayered film entrapping the drug molecules. When water
is later added, large vesicles encapsulating the drug molecules
are produced.
2.2.6. pH jumping
pH jumping is a quick liposome preparation method which also
circumvents the use of organic solvents. When a solution of phos-
phatidic acid in water is subjected for a short time (<2 min) to a 3.5
fold increase in pH (from
3 to 10.5–11), SUVs are formed [33].
When the same method is applied to a mixture of phosphatidic
acid and phosphatidyl choline, it yields similar results with the
ratio of phosphatidic acid : phosphatidyl choline controlling the
percentage of SUVs versus LUVs obtained (i.e., the higher the ratio
of phosphatidic acid : phosphatidyl choline, the higher the percent-
age of SUVs obtained [33].
2.2.7. Hydration in a packed bed of colloidal particles
This method uses a packed bed of colloids to produce SUVs
(<100 nm in diameter) in a single step [34]. In this method, lipo-
somes are obtained when lipids and amphiphilic molecules dis-
solved in an organic solvent are dried on asymmetric alumina
particles packed in a capillary, then hydrated with an aqueous buf-
3
Advanced Drug Delivery Reviews 176 (2021) 113851
fer. Drugs desired to be encapsulated may be added to the hydrat-
ing buffer.
2.2.8. Freeze-thaw
Freeze-thaw cycles are typically incorporated into liposome
synthesis to improve lipid packing and formation of unilamellar
vesicles [35]. This technique may be used with any liposome
preparation method. For example, after thin film hydration the
solution can be sonicated at room temperature, frozen at
196 °C in liquid nitrogen and left at room temperature to melt.
As the liposomal solution thaws, vesicles fuse forming LUVs. Up
to 10 freeze-thaw cycles may be used to achieve the intended
results. Finally, if smaller vesicles are desired, the resulting solu-
tion may be sonicated again at room temperature. The freeze-
thaw technique is not optimal when using high concentrations of
lipid for synthesis [1]. Using this technique, a drug encapsulation
efficiency of 2030% was reported [1,36].
2.3. Methods of liposome characterization
The key aspects that define the efficacy of a liposome formula-
tion include size, zeta potential, encapsulation efficiency, release,
stability, and pharmacokinetics. Size and zeta potential are proper-
ties defined by the liposome preparation method and composition,
respectively. Drug encapsulation efficiency and stability are critical
to protect and deliver the drug payload. Inefficient encapsulation
can lead to significant waste of expensive drugs. Drug release is
desired in the site of interest; premature drug release may cause
undesirable ‘‘off-target” effects. The pharmacokinetics of the lipo-
some are described by the circulation time and biodistribution of
the drug delivery vehicle. Together, encapsulation, stability,
release, circulation time, and biodistribution characterize the abil-
ity of a drug delivery vehicle to achieve the goal of delivering the
active drug to the diseased site.
2.3.1. Drug encapsulation efficiency and release
The encapsulation efficiency is a measure of the amount of drug
incorporated into the liposome during formulation. It is defined by
subtracting the free non-incorporated drug from the total drug and
dividing by the total drug initially added. This can be determined
using different methods, depending on the chemistry of the drug.
The concentration of drug in solution may be determined spec-
trophotometrically, fluorometrically, or using radiologic methods
[1].
Characterization of drug release is often performed in vitro
using a dialysis method. Liposomes are placed inside pre-wetted
dialysis bags with a selected molecular weight cut-off to entrap
the liposomes but allow the drug to permeate across the mem-
brane. The concentration of drug released is measured at different
time points. This provides a measure of the rate the drug will be
released from liposome formulations [1]. In reality, drug release
in vivo may be impacted by dilution in the bloodstream, pH, blood
plasma proteins, cells, or turbulent flows.
2.3.2. Size, zeta potential, and stability
Liposomal stability is an important indicator of its potential effi-
cacy and utility in clinical use. Often, the stability of a formulation is
evaluated by performing physical assessments of the liposomes at
multiple timepoints (e.g., days, week, or months) and assessing
drug leakage and nanoparticle size. Undesirable changes in the
physical characteristics of a liposome formulation include aggrega-
tion of the particles and physical degradation of the lipid membrane
over time. Liposomal diameter and surface charge can be deter-
mined using dynamic light scattering (DLS) and phase analysis light
scattering (PALS), respectively. Liposomes with neutral surface
charge aggregate and are unstable for drug delivery applications.
D.E. Large, R.G. Abdelmessih, E.A. Fink et al. Advanced Drug Delivery Reviews 176 (2021) 113851

Fig. 1. Liposomes are composed of a diverse array of molecular components such as lipids, sterols, polysaccharides, and surfactants. Representative structures of these
molecules are shown.

Fig. 2. Incorporation of cholesterol and PEG affects liposomal drug encapsulation and release. (A) Incorporation of cholesterol decreases drug encapsulation within liposomes,
as evidenced by decreasing paclitaxel encapsulation with increasing incorporation of cholesterol. Reproduced with permission [49]. Copyright 2007, Taylor & Francis. (B)
PEGylation of liposomes decreases drug release relative to use of free drug or encapsulation within non-PEGylated liposomes. Reproduced with permission [73]. Copyright
2010, Dove Medical Press Limited.

An increase in size is used to assess the uniformity and stability of
the liposome population over time. Atomic force microscopy (AFM)
is useful for measuring liposome mechanical characteristics, such
as Young’s modulus. Fourier-transform infrared spectroscopy
(FTIR) is used to analyze the makeup of the lipid membrane and
4
ensure degradation has not occurred [37]. Lyophilization is utilized
to prolong shelf life by reducing lipid oxidation. These techniques
are implemented at various timepoints over the shelf-life of the
liposome formulation; large deviations from initial readings may
indicate instability of a given formulation.
D.E. Large, R.G. Abdelmessih, E.A. Fink et al. Advanced Drug Delivery Reviews 176 (2021) 113851

Fig. 3. Liposomal composition affects stability. (A) Cholesterol enhances liposomal structural stability, enabling maintenance of liposomal size throughout 10 weeks.
Reproduced with permission [49]. Copyright 2007, Taylor & Francis. (B) Lipids with higher gel-liquid phase transition temperature Tm, such as DSPC and DPPC, increase
liposomal stability and cargo retention relative to lipids of lower Tm, DMPC. Reproduced with permission [41]. Copyright 2004, Taylor & Francis.

Fig. 4. Liposomal composition affects surface charge and pharmacokinetics. (A) Cationic lipids DOTMA and DOTAP enable non-viral gene delivery, as demonstrated by
luciferase expression in the lungs (h), spleen ( ), heart ( ), liver (4), and kidneys ( ). Reproduced with permission [85]. Copyright 1997, Mary Ann Liebert, Inc. (B)
PEGylation of doxorubicin encapsulating liposomes (Doxil Ò) increases blood circulation time relative to administration of free drug, as demonstrated by differences in blood
concentrations of doxorubicin 24 h after infusion. Reproduced with permission [105]. Copyright 2003, Springer Nature.

5
D.E. Large, R.G. Abdelmessih, E.A. Fink et al.
Fig. 5. Liposomal composition affects stimuli response. (A) Liposomes composed of pH-responsive lipid DOPE become unstable in response to acidic pH (<7), denoted by the
increased release of cargo as pH decreases. Reproduced with permission [108]. Copyright 2001, Elsevier. (B) Temperature-responsive liposomes release doxorubicin when the
environmental temperature is greater than physiological temperature (37 °C), as evidenced by increased dox release at temperatures at or above 42 °C. Reproduced with
permission [110]. Copyright 2010, Elsevier.
2.3.3. Pharmacokinetics
Pharmacokinetics describe what happens to the liposome and
drug after injection into the body. Drug pharmacokinetics are influ-
enced by encapsulation within a liposome formulation. Liposome
pharmacokinetics are characterized by the rate of removal from
the bloodstream and accumulation in organs. Pharmacokinetic
studies are performed by taking blood draws from subjects
injected with the liposome formulation over a period of time. Lipo-
somes and drugs are often detected by either fluorescent or radio-
logic labeling. This information is frequently analyzed using the
pharmacokinetic two-compartment model, in which the body is
divided into central and peripheral compartments. The central
compartment is comprised of plasma and tissues where vesicle
distribution occurs rapidly, while the peripheral compartment is
made up of tissues where distribution happens slowly. Mathemat-
ical models are fit to each of these compartments to model and
track liposome dispersal and elimination. Because liposomes are
often comprised of components that exist in the human body,
tracking of adsorption, degradation, metabolism, and excretion is
relegated to natural processes.
3. Molecular components of liposomal drug delivery vehicles
The liposome composition may be derived from natural or syn-
thetic lipids, polysaccharides, sterols, or surfactants (Fig. 1).
Research in liposome formulation has led to the emergence of
unique properties, including: enhanced drug encapsulation, stim-
uli responsiveness, tissue-targeting, prolonged blood circulation,
reduced drug toxicity in non-target tissues, and diagnostic capabil-
ities (Figs. 2–5). Collectively, these features increase therapeutic
efficacy relative to administration of free drug in a variety of clin-
ical applications.
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Advanced Drug Delivery Reviews 176 (2021) 113851
3.1. Natural lipids
Lipids have a diverse repertoire of structures that share a hydro-
philic headgroup and hydrophobic, hydrocarbon tail. The head-
group may be negatively charged, positively charged, or
zwitterionic (having both a negative and positive charge, resulting
in net neutrality). The headgroup may also be chemically modified
to allow conjugation with other molecules. For example, phos-
phatidylethanolamine (PE) is often conjugated to polyethylene gly-
col via the amine group. The charge of the headgroup bestows
stability, as charged liposomes electrostatically repel each other.
The hydrophobic tails may vary in acyl chain length, be symmetric
or asymmetric, and be saturated or unsaturated. The backbone of
the lipid is often a phosphate, glycerol, or sphingosine group. These
distinguishing chemical features regulate bilayer assembly defined
by lipid packing, response to pH, stability, drug encapsulation and
release, and other liposome behaviors.
3.1.1. Phospholipids
Natural lipids, such as phosphatidylcholine (PC) and PE are the
most common phospholipids present in mammalian cell mem-
branes [38]. Glycerophospholipids, i.e. lipids with a glycerol back-
bone, are the predominant phospholipid in eukaryotic cells [39].
Egg phosphatidylcholine (EPC) and egg phosphatidylglycerol
(EPG) are derived from living tissues; they are composed of a mix-
ture of lipid components. As a consequence, there may be inconsis-
tencies in the amount of the components from batch to batch. For
example, EPC derived from chicken egg is composed of L-a-
phosphatidylcholine with acyl chain lengths varying from 14 to
22 and 0 to 4 degrees of saturation. Other natural lipids are derived
from living organisms and subsequently processed to yield a nearly
pure lipid composition. In addition, lipid composition may be
D.E. Large, R.G. Abdelmessih, E.A. Fink et al.
defined by the degree of saturation or unsaturation of the acyl
chain. Liposomes composed of PCs with long, saturated acyl chains
(high Tm) have demonstrated greater stability in vivo compared to
formulations with PCs with short, unsaturated acyl chains (low Tm)
[40,41]. Vesicles composed of endogenous lipids are inherently
biocompatible and ideal for therapeutic applications as they mimic
native cell membranes.
3.1.2. Sphingolipids
Sphingolipids are another type of natural lipid found predomi-
nantly in mammalian cell membranes. Sphingolipids are com-
prised of an 18-carbon amino-alcohol sphingosine back bone;
they play a critical role in cell membrane structure and as regula-
tory signaling molecules [42]. The incorporation of sphingolipids,
such as sphingomyelin (SM), in liposomal formulations exhibit
increased stability in vivo, longer blood circulation, and increased
therapeutic efficacy [43]. Ceramides are a type of sphingolipid con-
sisting of a sphingosine and a single fatty acid. The incorporation of
ceramides in liposomal formulations increases membrane fluidity
and fusion capabilities with dermal cells [44].
3.1.3. Sterols
Sterols are a class of natural lipid molecules present in nearly all
living organisms [45]. There are three subtypes of sterols: phytos-
terols zoosterols, and mycosterols, which are found in plants, ani-
mals, and microorganisms, respectively [45]. Cholesterol is an
endogenous amphiphilic zoosterol and is a critical component of
mammalian cell membranes. Within the cell membrane, choles-
terol is primarily confined to lipid rafts and plays an important role
in processes of regulating membrane integrity and lipid raft func-
tionality [46]. The incorporation of cholesterol within liposomal
formulations has demonstrated increased stability in vivo and
decreased leakiness across the lipid bilayer, resulting in the pro-
longed and controlled release of cargo [37,47,48]. The incorpora-
tion of 20–50 mol% cholesterol in liposomal formulations
demonstrated reduced encapsulation efficiency [49] and increased
stability in vivo [47], relative to controls lacking cholesterol.
Cholesterol rich liposomes remained stable in the blood stream
for over 6 h, while cholesterol free liposomes were only stable
for a few minutes [47].
3.1.4. Polysaccharides
Polysaccharides are long-chain polymeric carbohydrates con-
sisting of monosaccharides bound by glycosidic linkages. Polysac-
charides play an important role in cellular communication and
are present in cell membranes to aid in processes of cell and tissue
recognition, as well as certain transport mechanisms [50]. Coating
the lipid membrane with oligo- or polysaccharides may target lipo-
somes to cell receptors or extend circulation [51,52]. Polysaccha-
rides have additional properties that make them well suited for
use in drug delivery systems, such as biocompatibility and anti-
viral, anti-bacterial, and anti-tumoral properties [53]. Mucosal sur-
faces of the body are particularly good targets for polysaccharide
recognition, thus nasal, pulmonary, peroral, and gastrointestinal
epithelium routes are heavily investigated for targeting with
polysaccharide-coated liposomes [53]. Popular polysaccharides
used in liposomal formulations are chitosan [54] and hyaluronan
[55,56].
3.2. Synthetic lipids
Synthetic lipids, lipids not occurring naturally or derived from
living sources, are commercially synthesized and frequently utilized
as components of therapeutic liposomes. Though these materials are
not endogenous, they are biologically and structurally similar to
natural lipids, exhibit high biocompatibility, and are synthesized
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Advanced Drug Delivery Reviews 176 (2021) 113851
with high purity. Commonly utilized synthetic lipids include phos-
phatidylcholines, phosphatidylethanolamines, and phosphatidyl-
glycerols, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
and dioleoyl phosphatidylglycerol (DOPG), respectively. Synthetic
lipids are often chosen over natural lipids due to their purity, com-
mercial availability, chemical functionality, and cost effectiveness.
3.3. Surfactants
Surfactants are classified as molecules that reduce the surface
tension of the liquid in which it is incorporated. Surfactants, com-
monly referred to as edge activators, are useful additives in lipo-
some formulations. Edge activators are typically single acyl-chain
surfactants that serve to destabilize the lipid bilayer of liposomal
nanoparticles, thereby increasing vessel deformability [57]. Edge
activators were shown to increase the dermal penetrative ability
of liposomes in anti-cancer [58,59], anti-fungal [60], and transder-
mal applications [61]. Frequently utilized edge activators include:
sodium cholate, Span 60, Span 80, Tween 60, and Tween 80. The
charge of edge activators may also be utilized to increase therapeu-
tic efficacy. For example, using sodium cholate, with a positive zeta
potential, electrostatically binds with negatively charged compo-
nents like DNA. Surfactant type and concentration may be lever-
aged to improve the therapeutic efficiency of liposomal drug and
gene delivery [62,63].
4. Effect of composition on liposome characteristics &
functionality
4.1. Drug encapsulation
Traditional therapeutic compounds, despite in vitro effective-
ness, are often limited in vivo by poor pharmacokinetic properties,
rapid clearance, low plasma solubility, or poor biodistribution.
Liposomal drug delivery is designed to circumvent these limita-
tions by encapsulating the drug of interest inside a vehicle that
demonstrates favorable in vivo stability, pharmacokinetics, and
biodistribution. Achieving high encapsulation efficiency is another
critical parameter of liposomal drug delivery success. While there
are various techniques employed to increase encapsulation effi-
ciency, it can be particularly difficult to efficiently load a drug into
small liposomes (50–150 nm) due to their low entrapment vol-
umes. This challenge is addressed through the use of reverse phase
evaporation and freeze-thaw cycling [64,65]. Hydrophobic and
hydrophilic compounds may be easily loaded into liposomes with
high encapsulation efficiency via association with the lipid bilayer
or aqueous core, respectively.
Active loading is a drug encapsulation method that converts the
drug from membrane permeable to impermeable. For example, a
trans-membrane gradient is introduced in order to drive drug
molecules into empty vesicles via a pH gradient. It was found that
generating pH gradients by using ammonium sulfate [66] or citrate
buffer [67] improve the encapsulation efficiency of amphiphilic
drugs while maintaining a simple, economical, and safe process
[68]. These processes are used commercially in the drug loading
of many FDA approved liposomal systems, such as DoxilÒ, Myo-
cetTM, and DuanoXomeÓ.
The physicochemical properties of liposomes may affect the
encapsulation efficiency during preparation, including: size, sur-
face charge, composition, and surface modifications [69]. Incorpo-
ration of long acyl chain lipids were shown to increase
encapsulation efficiency of hydrophobic drugs within the bilayer
and improved drug retention [70]. Incorporation of cholesterol
D.E. Large, R.G. Abdelmessih, E.A. Fink et al.
can negatively impact the encapsulation efficiency for drugs that
are incorporated into the lipid membrane [71].
The liposome formulation may be tailored to control the drug
release rate, leading to improved therapeutic efficacy of the drug.
Aspects of liposomal formulation that may be optimized to effect
drug release include: zeta potential, incorporation of cholesterol,
and the unique characteristics of the constitutive lipids (head
groups, acyl chain length, degree of saturation, and phase transi-
tion temperature) [71].
Characteristics of the lipids in the liposome formulation affect
packing of the lipid bilayer, which may be optimized to achieve
sustained drug release. Increasing the degrees of unsaturation of
the acyl chain has demonstrated increased drug release due to
the leakiness of the lipid bilayer [72]. Liposomes made with 100%
DOPC exhibited fast drug release, with no further release after
24 h, whereas liposomes made with 100% DSPC showed slow, sus-
tained release that lasted seven days [72]. A formulation made
with a mixture of the two showed an intermediate release profile,
showing how mixtures of lipids can serve to tune drug release.
Liposomal incorporation of polyethylene glycol (PEG) has also
demonstrated decreased drug release [73]. A higher degree of lipid
saturation (and higher Tm), affects the fluidity of the lipid mem-
brane. Increasing the Tm decreases overall drug release [71].
4.2. Stability
Liposomal stability may be classified into physical, chemical,
and biological. Physical and chemical stability often refer to the
ability of the liposomal formulation to maintain its properties over
time. Phospholipids are prone to several chemical degradation
reactions, including hydrolysis at ester bonds and peroxidation of
unsaturated acyl chains; these phenomena can affect the long-
term stability of a liposome formulation. Stability may also
describe the ability of a liposome formulation to be reconstituted
from lyophilization. Various excipients are used to balance osmo-
lality, pressure of the interior and exterior of the liposome. Biolog-
ical stability refers to liposome integrity in the presence of serum
proteins. Upon entering the bloodstream, liposomes bind serum
proteins, a phenomenon known as opsonization, which results in
rapid clearance [74]. The avoidance of liposomal opsonization
and subsequent increase in blood circulation time can be achieved
through the incorporation of PEG, as described in section 4.4. The
addition of cholesterol in liposomal formulations, alone or in con-
junction with PEG, also increases blood circulation time [75,76].
Overall, stability describes the maintenance of the initial liposome
properties in response to different stresses.
4.3. Surface charge
Each phospholipid exhibits a net charge as a function of their
headgroup chemistry and solution pH. The liposome compositions
in the final formulation impacts the overall zeta potential of the
liposome, which affects the function and efficacy of the vesicles
as a therapeutic delivery system.
Anionic liposomes, i.e. those with negative surface charge, are
composed of lipids with negatively charged head groups, such as:
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 1,2-
dioleoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (DOPG). Anionic
liposomes are utilized for DNA transfection [77]. The incorporation
of negatively charged lipids in liposomal formulations also
enhanced the delivery of cardiotoxic drugs, such as doxorubicin,
reducing systemic toxicity compared to administration of free drug
[78]. Further, anionic liposomes demonstrated enhanced vascular
extravasation and lower accumulation in the vascular endothe-
lium, relative to cationic liposomes [79]. Due to the electrostatic
repulsion of anionic liposomes with the negatively charged surface
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Advanced Drug Delivery Reviews 176 (2021) 113851
of cell membranes, nonspecific cell uptake is reduced compared to
that of cationic liposomes. Reduced nonspecific uptake also
enables anionic liposomes to circulate in the bloodstream longer
than cationic liposomes [80].
Liposomes with positive surface charge can be synthesized via
the incorporation of positively charged lipids. The most commonly
utilized lipids in cationic liposomes include: 1,2-dioleoyl-3-trime
thylammonium-propane (DOTAP), C-Chol, N-(1-(2,3-dimyristyloxy
propyl)-N,N-dimethyl-(2-hydroxyethyl) ammonium bromide
(DMRIE), 1,2-dimyristyloxypropyl-3-dimethyl-hydoxyethylammo
nium bromide (DODAC), and 1,2-di-O-octadecenyl-3-trimethylam
monium propane (DOTMA). Cationic liposomes are noted for their
usefulness in nonviral gene transfection and siRNA delivery [81–
85]. Positively charged liposomes are utilized in the formulation
of liposomal vaccine adjuvants [86] as they possess inherent
immunostimulatory properties [87,88]. Augmented immunogenic
response and increased vaccine efficiency was demonstrated with
cationic liposomal vaccines [89]. Further, cationic liposomes have
demonstrated enhanced peritoneal retention compared to neutral
or anionic liposomes, making them ideal drug carriers for the treat-
ment of gastrointestinal and gynecological malignancies [90].
However, due to electrostatic interactions between the
positively-charged lipid membrane of the vesicle and negatively-
charged cell membranes, cationic liposomes are nonspecifically
internalized by cells to a greater extent than anionic liposomes.
4.4. Pharmacokinetics
Differences in liposomal composition can alter pharmacokinetic
parameters in vivo, as noted by changes in blood clearance rate,
half-life, and biodistribution. Liposomal formulations which pro-
long blood circulation enable higher accumulation and deposition
of therapeutic cargo within target tissues.
4.4.1. Clearance rate and half-life
Liposomal blood clearance rate and half-life are greatly
impacted by liposomal composition. The mononuclear phagocyte
system (MPS), which consists of tissue resident macrophages and
blood monocytes [91], is primarily responsible for the clearance
of liposomes from the blood stream. Circulating neutrophils also
significantly hinder drug delivery [92]. There is considerable
research devoted to modulating liposomal composition to reduce
uptake by the MPS.
Liposomal opsonization upon systemic administration directs
liposomes to interact with receptors on the surface of liver and
spleen resident macrophages of the MPS, subsequently inducing
internalization and degradation. In a mechanistically analogous
fashion, liposomal opsonization may also direct particles for
uptake in the liver by hepatocytes [93]. The incorporation of PEG
in liposome formulations, known as ‘‘stealth” liposomes, has pro-
longed the circulation time and subsequently increased liposomal
half-life by hindering opsonization or preferentially binding pro-
teins that evade uptake [94,95] Liposomal PEGylation imparts a
greater surface hydrophilicity as well as a barrier of steric hinder-
ance, both of which affect the binding of serum proteins to the
liposome surface [96,97]. Further, liposomes composed primarily
of neutral phospholipids with a small molar percent of negatively
charged glycolipid, principally monosialoganglioside (GM1), have
also demonstrated prolonged circulation in vivo. Mechanistically,
GM1 reduces liposomal opsonization through steric hinderances
introduced by the negatively charged sialic acid and the physical
boundary provided by the carbohydrate chains [98].
4.4.2. Biodistribution
Liposomal biodistribution, i.e. the dispersion of liposomes
throughout the body (often the lungs, kidneys, liver, spleen, stom-
D.E. Large, R.G. Abdelmessih, E.A. Fink et al.
ach, intestines, and brain) can be altered through the incorporation
of different molecular components.
Liposomes have garnered attention in the field of cancer drug
delivery for the ability to deliver toxic drugs to cancer cells. Accu-
mulation of liposomes within a tumor is facilitated by the
enhanced permeability and retention (EPR) effect. Though not fully
understood, the EPR effect is hypothesized to result from leaky vas-
culature and an impaired lymphatic system present in tumors.
Tumor accumulation via the EPR effect is enhanced by liposomes
with prolonged blood circulation. Other factors which have
impacted tumor accumulation include: liposome size [99], elastic-
ity [100], and surface charge [79,101]. Liposomal elasticity is mod-
ified via the incorporation of cholesterol, surfactants, and lipids of
varying acyl chain length and degree of saturation. Liposomes
exhibiting low to moderate Young’s Moduli (~0.045–28 MPa) accu-
mulated significantly more in tumors than high Young’s modulus
liposomes [100,102]. Modulation of liposomal surface charge
demonstrated enhanced accumulation of anionic liposomes within
tumors, relative to their cationic or neutral counterparts [103].
Liposomal surface modifications, such as the conjugation of pep-
tide or antibodies to enable targeting of liposomes to overex-
pressed receptors on diseased cells, have also been investigated
as a method to increase tumor accumulation (reviewed here
[104]).
Liposomes predominantly accumulate in the organs of the renal
and lymphatic systems, such as the liver, kidneys, and spleen,
respectively. Liposomal accumulation within the liver and spleen
is modulated by cells of the MPS present within these organs. Renal
accumulation and clearance of liposomes by the kidneys occurs
through physiological processes of blood filtration and waste
excretion. Accumulation within other organ systems, such as the
respiratory, nervous, and cardiovascular system, i.e. the lungs,
brain, and heart, respectively, is generally minimal. Researchers
have taken advantage of these pathways to deliver anti-fungal
agents directly to the kidney (amphoteric B).
5. Liposomal composition imparts unique functionalities
The advent of stimuli-responsive liposomes has accelerated the
use of nanoparticles for site-specific therapeutic delivery. Environ-
mentally specific stimuli, such as pH, can cause destabilization of
the liposomal membrane, enabling local deposition of drug pay-
load in target tissue. External stimuli have also been explored,
including heat and light. By utilizing an external stimulus, the site
of release can be controlled as well as the rate of release. Stimuli-
responsive liposomes exhibited increased efficacy in anti-tumor
and gene delivery due to the distinctive properties of the tumor
microenvironment, including pH and enzymatic activity. By lever-
aging tumor specific environmental cues, the drug is delivered to
the tumor site in a way that is both targeted and efficient [106].
5.1. pH responsive liposomes
pH-sensitive liposomes were explored for their ability to target
tumors and regions of inflammation. When pH-responsive lipo-
somes encounter acidic conditions, components of the lipid bilayer
become protonated. This chemical change subsequently disrupts
the structural integrity of the bilayer, causing the membrane to
rupture. Thus, when pH-responsive liposomes come into contact
with an acidic microenvironment, such as the endosome of a cell,
they release drug payload [107,108]. pH-sensitive liposomes may
be optimized to maintain necessary characteristics of liposomes,
such as long circulation time, while optimizing their ability to
destabilize and deposit their payload effectively at a desirable
pH. The incorporation of lipids with high phase transition
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Advanced Drug Delivery Reviews 176 (2021) 113851
temperatures, such as DSPC, soy PC, and HSPC, and additives such
as cholesterol and PEG were shown to be successful in maintaining
the circulation time of liposomes. Traditionally, PE was utilized in
the synthesis of pH-sensitive liposomes to stabilize the carriers at
physiological pH. More recently, alternative mechanisms such as
the incorporation of pH-sensitive and fusogenic molecules were
explored [109].
5.2. Temperature responsive liposomes
Temperature-sensitive liposomes deposit their payload when
external heat is applied, but the modes by which the membrane
is destabilized differ. Traditional temperature-sensitive liposomes
are composed of lipids that have a gel-to-liquid phase transition
temperature or Tm that is higher than the body temperature, typi-
cally ~42 °C. Recently, temperature-sensitive polymers or lysoli-
pids were used to initiate membrane destabilization, followed by
payload deposition when heated [110]. Above the Tm, the lipid
membrane increases fluidity and permeability to allow for maxi-
mum drug release. Traditional thermo-sensitive liposomes were
formulated using a mixture of lipids with different phase transition
temperatures which increases lipid packing disorder, subsequently
increasing cargo permeability. However, the high thermal dose
threshold for thermo-responsive liposomes may cause damage to
healthy tissues during heat application. To address this limitation,
a new generation of thermo-sensitive liposomes was explored,
using temperature-sensitive polymers and lysolipids to lower the
phase transition temperature, while still enabling rapid drug
release [111].
5.3. Theranostic liposomes
Theranostic liposomes, incorporating a diagnostic (i.e. imaging
modality) and therapeutic agent, are multifunctional nanomedici-
nes for disease treatment and monitoring [112]. Theranostic lipo-
somes can be formulated to be amenable to several imaging
techniques, including magnetic resonance (MRI), near-infrared flu-
orescent (NIR), and nuclear imaging [113,114].
6. Liposomal drugs in clinical use and preclinical development
To emphasize the versatility of liposomes as drug carriers, we
will discuss a few illustrative examples of different liposomal drug
formulations either approved by the U.S. Food and Drug Adminis-
tration (F.D.A.) or the European Medicines Agency (E.M.A.) for clin-
ical use, or in the clinical trials stage. Table 1 lists all liposomal
drug formulations currently approved for clinical use. Liposomes
are used as carriers for analgesic medications such as morphine
and fentanyl, and anesthetics such as ropivacaine [115]. They are
also used as vehicles for drugs that prevent and treat a broad spec-
trum of diseases such as fungal (e.g., amphotericin B and nystatin),
bacterial (e.g., amikacin and doxycycline) and viral infections, as
well as chemotherapeutics used for treatment of different types
of cancers (e.g., camptothecin, cisplatin, daunorubicin, docetaxel,
doxorubicin, gemcitabine, oxaliplatin, paclitaxel, and vincristine)
[115–117].
6.1. Anti-cancer
Aroplatin is currently in phase II clinical trials and is being
investigated for the treatment of metastatic colorectal carcinoma
through intrapleural injection [118]. Colorectal carcinoma is a
malignant tumor growth in the epithelium that is part of the
mucosa layer of the large intestine [119]. Aroplatin contains a
cis-bis-neodecanoato-trans-R, R-1,2-diaminocyclohexane platinum
D.E. Large, R.G. Abdelmessih, E.A. Fink et al. Advanced Drug Delivery Reviews 176 (2021) 113851

Table 1
Liposomal drug formulations approved for clinical use.

Name Company Liposomal Composition Drug Encapsulated Drug Type Route of Clinical References
(molar ratio) Administration Approval
Year
Abelcet Leadiant Biosciences, DMPC : DMPG Amphotericin B Antifungal I.V. 1995 [115,117,132,133]
Inc. (2.3 : 1)
Ambisome Fujisawa Healthcare, HSPC : DSPG : Cholesterol : Amphotericin B Antifungal I.V. 1997 [117,158]
Inc. and Gilead Sciences, Amphotericin B
Inc. (5 : 2 : 2.5 : 1)
Amphocil Zeneca Pharmaceuticals Cholesteryl sulphate : Amphotericin B Antifungal I.V. 1993 [115,159]
Amphotericin B
(1 : 1)
Amphotec Sequus Pharmaceuticals Cholesteryl sulphate : Amphotericin B Antifungal I.V. 1996 [116,117,160]
Inc. Amphotericin B
(1 : 1)
Arikayce Insmed, Inc. of DPPC and Cholesterol : Amikacin Antibacterial Oral Inhalation 2018 [142,146,161]
Bridgewater, NJ. Amphotericin B
(0.6–0.79 : 1 wt ratio)
DaunoXome Galen US, Inc. DSPC : Cholesterol Daunorubicin Chemotherapeutic I.V. 1996 [117,162]
(2 : 1)
DepoDur Pacira Pharmaceuticals, DOPC : DPPG : Cholesterol : Morphine sulfate Narcotic Analgesic Epidural 2004 [117,139]
Inc. Tricaprylin and Triolein
(507 : 11 : 76 : 6 : 1)
Doxil Johnson & Johnson HSPC : Cholesterol : DSPE- Doxorubicin Chemotherapeutic I.V. 1995 [117,135]
PEG2000
(11.2 : 7.8 : 1)
Epaxal Johnson & Johnson DOPC : DOPE Hepatitis A virus Vaccine I.M. 1993 [117,163]
(3 : 1) antigen, strain RG-
SB
Exparel Pacira Pharmaceuticals, DEPC : DPPG : Cholesterol : Bupivacaine Anesthetic I.V. 2011 [117,164]
Inc. Tricaprylin
(7.6 : 1 : 10 : 3.5)
Evacet Liposome Company Inc. (Hydro Soy PC, cholesterol Doxorubicin Chemotherapeutic I.V. 1995 [115,133,165,166]
and DSPE-PEG) :
Doxorubicin
(8 : 1)
Inflexal V Johnson & Johnson 70% Lecithin, 20% Cephalin Influenza virus Vaccine I.M. 1997 [117,151]
and 10% Phospholipids antigen, strains A
(DOPC : DOPE, 3 : 1) and B
Lipodox Sun Pharmaceutical DSPC : Cholesterol : DSPE- Doxorubicin Chemotherapeutic I.V. 1995 [167–170]
Industries Ltd. PEG2000
(10.9 : 7.3 : 1)
Marqibo Acrotech Biopharma, Sphingomyelin : Vincristine Chemotherapeutic I.V. 2012 [117,171]
LLC Cholesterol
(1.5 : 1)
Mepact Takeda Pharmaceutical DOPS : POPC Mifamurtide Immunomodulator/ I.V. 2004 [117,172]
Limited (1 : 2.3) Antitumor
Myocet Zeneus Pharma Ltd. EPG : Cholesterol Doxorubicin Chemotherapeutic I.V. 2000 [117,173]
(1.2 : 1)
Onivyde Merrimack DSPC : MPEG-2000 : DSPE Irinotecan Chemotherapeutic I.V. 2015 [117,174,175]
Pharmaceuticals, Inc. (200 : 133.3 : 1)
Visudyne Novartis International Verteporfin : DMPC and Verteporfin Photosensitizer I.V. 2000 [117,176]
AG EPG
(1 : 8)

(II) (NDDP), a molecule structurally analogous to oxaliplatin. Plat-
inum coordination complexes inhibit the replication of DNA in
tumor cells by cross-linking the two DNA strands, inducing cell
death [117,120–123]. Aroplatin consists of MLVs encapsulating
NDDP, made of 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(10 -rac-
glycerol) (DMPG). The drug-loaded liposomes increase NDDP accu-
mulation in tumor cells, and reduce its side effects compared with
the unencapsulated form of the drug [117,124].
Depocyt is a cytarabine liposomal injection that is F.D.A.
approved for treatment of neoplastic meningitis through spinal
injection [116,125–127]. Neoplastic meningitis or ‘leptomeningeal
metastasis’ is inflammation of the brain caused by tumor growth in
the arachnoid mater and/or the pia mater of the meninges which
surround the brain and spinal cord [128]. As the disease pro-
gresses, tumor cells spread through the cerebrospinal fluid result-
ing in poor prognosis. DepoCyt contains the chemotherapeutic
cytarabine. The liposome in this drug formulation is composed of
cholesterol, triolein, DOPC and 1,2-dipalmitoyl-sn-glycero-3-
phospho-(10 -rac-glycerol) (DPPG) in a molar ratio of 11:1:7:1
[116]. The encapsulated drug is slowly released from the 3–
30 mm MLVs – with high internal volume to surface area ratio.
These giant vesicles consist of 96% aqueous foam and 4% lipids
[117,129]. The liposome encapsulated cytarabine was shown to
be more effective in targeting and destroying tumor cells in the
meninges and cerebrospinal fluid compared with other forms of
the drug [117,130]. In 2017, Pacira BioSciences, Inc. discontinued
the production of Depocyt due to manufacturing difficulties [131].
Doxil is the first F.D.A. approved drug containing PEGylated
liposomes [105,117,132–135]. It is prescribed for the treatment
of different types of cancers including ovarian cancer,
AIDS-related Kaposi sarcoma, and multiple myeloma [117,136].
The chemotherapeutic encapsulated in Doxil is doxorubicin
HCl (Dox). The liposomes in Doxil are composed of

10
D.E. Large, R.G. Abdelmessih, E.A. Fink et al.
L-a-phosphatidylcholine hydrogenated soy (HSPC), cholesterol and
N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-
glycero-3-phosphoethanolamine sodium salt (MPEG-DSPE) in a
molar ratio of 56:38:5 [117,137]. The SUVs are 80–100 nm
[117,135], and are comprised of Dox:lipid with a mass ratio of
8:1 [117,138]. This high drug:lipid ratio is achieved by a remote
loading technique using an ammonium sulfate gradient
[105,117,135]. Doxil showed an increased circulation time with a
half-life of 20–35 h and lower cardiotoxicity compared with the
free form of the drug [105].
6.2. Pain management
DepoDur is a morphine sulfate extended-release liposome
injection F.D.A. approved for treatment of severe pain [139]. The
liposomal composition of DepoDur consists of DOPC, DPPG, choles-
terol, tricaprylin and triolein in a mass ratio of 42:9:33:3:1, form-
ing 17–23 mm MLVs; the drug encapsulated is an analgesic,
morphine sulphate [117,140]. Morphine sulphate binds opioid
receptors, inhibitory G protein-coupled receptors, in the central
nervous system and mitigates severe pain [141,142]. Using the
morphine encapsulated liposomes, clinical trials demonstrated
consistent release of the drug up to 48 h post injection [143].
6.3. Anti-bacterial
In 2018, Arikayce, Amikacin Liposome Inhalation Suspension
(ALIS), was approved by the F.D.A. for the treatment of a bacterial
lung infection, mycobacterium avium complex (MAC) [144–147].
Two types of nontuberculous mycobacteria bacteria can cause
MAC: Mycobacterium avium and Mycobacterium intracellulare,
which mainly affect immunocompromised individuals. [148]. Ari-
kayce contains amikacin [149], a bactericidal aminoglycoside,
effective in vivo against different species of gram-negative bacteria
including Acinetobacter, Enterobacter, Klebsiella Pseudomonas, Pro-
teus, Serratia and Escherichia coli, and one species of gram-
positive bacteria, Staphylococcus [150]. Arikayce is the first liposo-
mal formulation approved to be administerd by inhalation. In this
formulation, the liposomal vehicle, which protects and stabilizes
the active drug until it reaches the lungs, is made of a phospholipid,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and choles-
terol in 2:1 mass ratio, while the concentrations of amikacin and
total lipids in the formulation are 70 mg/ml and 47 mg/mL, respec-
tively [116,147]. The innovative approach of this drug formulation
is that it overcame the difficulty of treating intracellular bacterial
infection by delivering the active drug directly into cells via lipo-
somes that are taken up by macrophages within the lungs [147].
In vivo studies in rats demonstrated a 5- to 8-fold increase in the
concentration of amikacin in pulmonary macrophages at 2, 6,
and 24 h after treatment, compared with the inhaled free form of
the drug [147].
6.4. Vaccines
Inflexal V, an inactivated trivalent influenza vaccine, is com-
prised of 150 nm unilamellar virosomes – liposomes whose sur-
faces are decorated with viral antigens, in this case,
haemagglutinin and neuraminidase glycoproteins of influenza
virus strains A and B [117,151–153]. The liposomes are composed
of 70% lecithin, 20% cephalin and 10% phospholipids (DOPC:DOPE,
75:25 molar ratio), where cephalin functions as a structural com-
ponent and a stimulant to B lymphocytes [117,154]. Each dose of
Inflexal V contains 15 mg of haemagglutinin-decorated monovalent
virosomes from three different pools of influenza viruses (two of
strain A and one of strain B) [151,155,155]. Inflexal V has shown
11
Advanced Drug Delivery Reviews 176 (2021) 113851
significantly improved immunogenicity and tolerability over other
types of conventional influenza vaccines [117].
Finally, it should be noted that decades of research into engi-
neering and optimizing these drug delivery systems have enabled
the record-breaking fast development of two vaccines, by Pfizer-
BioNTech and Moderna, against SARS-CoV-2 [156]. These vaccines
contain mRNA fragments of viral spike proteins – which enable
SARS-CoV-2 to get attached, and gain entry into cells – encapsu-
lated into lipid nanoparticles. The function of the lipid vesicles is
to protect the genetic materials from being degraded by enzymes,
and enable their uptake into cells, so the encoded viral proteins can
be synthesized by cellular machinery and an immune response is
initiated [157].
7. Conclusion
Liposome composition imparts unique characteristics and func-
tionality, making liposomes ideal carriers for a wide range of ther-
apeutic cargo and clinical applications. Since their discovery nearly
55 years ago, liposomes have become a staple in the field of drug
delivery. The use of liposomes in clinical applications has dramat-
ically reduced the off-target toxicity of various drugs, enabled pro-
longed blood circulation and beneficial drug biodistribution.
Most recently, liposomes have taken center stage in the battle
against SARS-CoV-2. By providing protection of genetic material,
lipid nanoparticles have enabled the successful translation of
mRNA vaccines and ushered in a new era in gene therapy. Given
the significant efficacy demonstrated by these vaccines, it is evi-
dent that liposomal drug delivery is paving the way for future suc-
cess in the delivery of genetic materials for a range of therapeutic
needs. As with the development of the SARS-CoV-2 mRNA vacci-
nes, optimization of lipid composition will play a critical role in
the development of these gene therapy-based treatments.
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