BioNanoScience (2022) 12:274–291

https://doi.org/10.1007/s12668-022-00941-x

A Comprehensive Review on Novel Liposomal Methodologies,

Commercial Formulations, Clinical Trials and Patents
Veera Venkata Satya Naga Lakshmi Andra1 · S. V. N. Pammi2 · Lakshmi Venkata Krishna Priya Bhatraju1 ·
Lakshmi Kalyani Ruddaraju1

Accepted: 12 January 2022 / Published online: 26 January 2022

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Liposomes are well-recognized and essential nano-sized drug delivery systems. Liposomes are phospholipid vesicles com-
prised of cell membrane components and have been employed as artificial cell models to mimic structure and functions of
cells and are of immense use in various biological analyses. Liposomes acquire great advantages and provide wide range
of applications as useful drug carriers in pre-clinical and clinical trials. This review summarizes exclusively on scalable
techniques for liposome preparation and focuses on the strengths and limitations with respect to industrial applicability.
Also, this review discusses the updated recent advancements in biomedical applications with a mention of key highlights of
commercially available formulations, clinical trials and patents in recent past. Furthermore, this review also provides brief
information of the classification, composition and characterization of liposomes.

Keywords Biological analysis · Drug delivery systems · Liposomes · Recent advancements

Abbreviations DELOS Depressurization of an expanded liquid

SUVs Small unilamellar vesicles organic solution suspension method
LUVs Large unilamellar vesicles SAS Super critical anti-solvent
GUVs Giant unilamellar vesicles SEM Scanning electron microscopy
MLVs Multi lamellar vesicles TEM Transmission electron microscopy
OLVs Oligo lamellar vesicles HPLC High-performance liquid chromatography
MVVs Multivesicular Vesicles HPTLC High-performance thin layer
LMVs Large multilamellar vesicles chromatography
SMVs Small multilamellar vesicles MRI Magnetic resonance imaging
PEG Polyethylene glycol DOX Doxycycline
W/O emulsion Water in oil emulsion VEGF Vascular endothelial growth factor
SCF Super critical fluid
DAC Dual asymmetric centrifugation
CAS Continuous anti-solvent method 1 Introduction
SRCPE Supercritical ­CO2 reverse phase evapora-
tion process Liposomes are spherical, closed structures composed of
RESS Rapid expansion of supercritical solution phospholipids in the colloidal size range of 5–200 nm and
contains one or more concentric/non-concentric mem-
branes, of around 4 mm thickness [1]. The liposomes con-
* Lakshmi Kalyani Ruddaraju sist of amphiphilic phospholipids with hydrophilic head and
lakshmikalyani.r@svcp.edu.in
hydrophobic tail, which aids in unique characteristics such
1
Department of Pharmaceutical Sciences, Shri Vishnu as self-sealing of liposomes in aqueous media. In recent past,
College of Pharmacy, Vishnupur, Bhimavaram 534201, much research has been focused on the delivery of antibiot-
Andhra Pradesh, India ics [2, 3], genes [4, 5], antifungal [6, 7], anti-inflammatory
2
Department of Basic Sciences & Humanities, GMR Institute [8, 9] and anti-cancer drugs [10, 11] and also used in many
of Technology (GMRIT), GMR Nagar, Rajam 532127, pharmaceutical, biological and medical fields.
Andhra Pradesh, India

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In 1965, A.D. Bangham first discovered that phospholipid 2 Classification

molecules can instantly form a closed bilayer vesicles in
aqueous media due to amphiphilic nature of liposomes [12]. Liposomes classified into unilamellar, multilamellar, oli-
Shortly, liposomes in a size range of 5–200 nm have been golamellar, and multi vesicular vesicles based on the num-
noticed for encapsulation of hydrophilic or hydrophobic ber of phospholipid bilayers, as shown in Fig. 1. Various
drugs into aqueous phase/bilayer membrane, which made a types of liposomes along with their particle sizes are given
pavement of liposomes for drug delivery applications. Soon in Table 1. The desirable size of liposomes for drug delivery
after first liposome-encapsulating drug reached clinical trials applications ranges from 50 to 200 nm [20, 21].
in 1985 [13], more than 40 liposome based formulations for Liposome size is the major factor for efficient delivery
drug delivery applications have been successfully entered of drugs into the body. The size of liposomes shows signifi-
into market from various clinical stages. cant effect on the pharmacokinetics of liposomes and drugs
A significant advantage of systemic liposomes as drug encapsulated into the liposomes. The size of liposomes less
formulations is high biocompatibility, low immunogenic- than 200 nm shows increased circulation and residence time
ity, biodegradability, increased efficiency, prolonged drug of liposomes in the blood, enhanced in vivo drug release
half-life, targeted delivery, lowered systemic toxicity and from liposomes and significant accumulation into the tumour
protection of sensitive molecules, with enhanced pharma- cells [22].
cokinetics. The utmost advantage of systemic liposomes
incorporation and release of two different materials with dif-
ferent solubility’s simultaneously [14]. Reports from various 3 Composition of Liposomes
investigations revealed different types of liposomes are clas-
sified based on number of bilayers, size and the liposomal 3.1 Phospholipids
composition and are discussed in further sections briefly.
Various publications are noted with focal point as conven- Liposomes are comprised of lipid and/or phospho-
tional methods [15, 16], biomedical applications [17, 18] and lipid molecules with different head groups and minimal
recent advances in liposomal methodologies in liposomal [19]. amounts of other additives which should be incorporated
In this review, we broadly focus on inventive ideas in methods in an internal aqueous core and an outer lipid bilayer. Phos-
of preparation and commercially available liposomal formu- pholipids are amphiphilic molecules, with a hydrophilic
lations with different routes of administration, characteristics phospholipid head and a hydrophobic fatty acid tail. Phos-
and their applications to overcome the hindrances with con- pholipids possess glycerophospholipids and sphingomyelins.
ventional preparations. In addition, this review discusses in a
broad range from conventional methods to recent advance-
ments in preparation techniques and new innovation technolo-
gies in liposomal preparation along with mechanism of forma- Table 1  Types of liposomes and their particle size
tion wherever possible with a mention of specific advantages
Types of liposomes Particle size Number
and limitations of each liposomal methodology. Further, ongo- of lamel-
ing research on clinical trials and patents sanctioned in recent lae
past is well detailed. Therefore, we anticipate this resource
Small unilamellar vesicles [SUVs] 20–100 nm 1
can give an overall pathway to choose an optimal method for
Large unilamellar vesicles [LUVs] > 100 nm 1
the researchers with an up to-date knowledge on various bio
Giant unilamellar vesicles [GUVs] > 1000 nm 1
medical applications with an idea on current research in clini-
Multi lamellar vesicles [MLVs] > 500 nm >5
cal trials and patents to make a pavement for liposomes from
Oligo lamellar vesicles [OLVs] 100-1000 nm 2–5
the pre-clinical research to production and clinical use.
Multi vesicular vesicles [MVVs] > 1000 nm 1

Fig. 1  Classification of
liposomes

SUV LUV MLV MVV

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Glycerophospholipids consists a lipophobic head and a lipo- vitamins, proteins and nucleic acids, which exists on the
philic tail. Different substitutions at head groups such as receptor surface of target cells.
phosphatidylcholine, phosphatidylinositol, phosphatidyl- Research on liposomal technologies was continuously
serine, phosphatidylethanolamine, phosphatidic acid, car- refined from conventional vesicles to “second-generation
diolipin, and phosphatidylglycerol result in various glycer- liposomes”, i.e. the extended-circulating liposomes with
ophospholipids [23]. Furthermore, the variation in length of controlled and gradual release of active pharmaceutical
the nonpolar moieties produces different glycerophospholip- ingredient, which can be achieved by modifying the phos-
ids, such as dipalmitoyl, dimyristoyl, or distearoylphosphati- pholipid composition, dimension and charge of the vesicle.
dylcholine. Additionally, the type of bonding, i.e. ether or Numerous particles like sialic acid or glycolipids [15, 33],
ester between glycerol and aliphatic chains, provides various unmodified dextrans and modified dextrans are used to
types of glycerophospholipids. To achieve charged vesicles, establish the modified surface liposomes [34].
charged phospholipids like stearyl amine and diethylphos-
phate can been used. Binding of sphingomyelin helps in
water permeability reduction and enhancement of proton 4 Liposome‑Based Carrier System
permeability in some kinds of liposomes [24]. Lipids are
capable to manipulate the surface charge, bio distribution, Liposomes are the drug carriers which are capable to carry
permeability, release and clearance of various formulations the drugs into specific target sites [16], and different carrier-
of liposomes [25]. The segments of phospholipids govern based systems are listed in Table 2.
the encapsulation efficiency, stability and toxicity of lipo-
somal formulations [26].
5 Methods of Preparation
3.2 Bilayer Excipients
Liposome preparation can be done by conventional methods
Phospholipids alone are usually inadequate for the prepara- such as Bangham method [thin film hydration], ether/ethanol
tion of liposomes, which might trigger defects in the bilayer injection method, reverse phase evaporation method, deter-
packing leading to outflow of captured drug due to unsatura- gent depletion method, heating method, microfluidic channel
tion of fatty alkyl chains or low phase transition tempera- method, membrane extrusion method, homogenization and
ture during storage. In order to avoid such outflow, various sonication method. Novel methods for liposomal-based drug
other bilayer excipients are added to the liposome composi- delivery involve freeze drying, dual asymmetric centrifu-
tion. Cholesterol and α-tocopherol are the most widely used gation [DAC] and supercritical fluid [SCF] methods since
excipients [27]. Modifications in the phospholipid bilayer a decade [46]. Depo-foam liposome technique, lysolipid
composition influence the liposome encapsulation efficiency. thermally sensitive liposome technique, non-PEGylated
The aggregates formed by electrostatic effect and the fluid- liposome technique and stealth liposome techniques are the
ity of phospholipid bilayers can be stabilized by using cho- innovative techniques used for the delivery of drugs in the
lesterol. The variation in the quantity of cholesterol used recent past, and each method and its status are discussed
in the liposome preparation is based upon the area of lipo- further.
some application. On the other side, α-tocopherol affords
for higher therapeutic potential through inducing reactive 5.1 Conventional Methods
oxygen species in injured tissue, thereby liposomes readily
ease the intracellular delivery and prolong the retention time 5.1.1 Thin Film Hydration Method [Bangham Method]
of encapsulated drugs [28].
The Bangham method is the first commonly used method
3.3 Additional Excipients for liposome preparation [12, 47]. This method utilizes an
organic solvent (dichloromethane, chloroform, ethanol and
Polyethylene glycol [PEG] on the liposome surface offers chloroform–methanol mixture) to dissolve lipids; further the
extended circulation property, protects the captured drug organic solvent can be removed by evaporation under vac-
from inactivation or metabolic degradation, further enhances uum at a temperature of 45–60ºC to form a thin lipid film.
stability and improves intracellular intake [29]. PEG may Subsequently, the thin lipid film gets hydrated in aqueous
produce stealth liposomes that are undetectable by the media by continuous agitation up to 2 h at a temperature of
body’s reticuloendothelial system [30, 31]. Moreover, PEG 60–70ºC where it swells to produce round closed liposomes
assists in decreasing particle’s aggregation and improves [48]. The schematic representation of Bangham method is
the stability on storage [32]. Cellular intake of PEGylated represented in Fig. 2.
liposomes can be enhanced by ligands such as antibodies, Advantages:

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Table 2  Different liposome carrier-based systems

Vesicle type Characteristics

Immunoliposomes • Aids in targeting the bioactive agents inside the body [35]
Virosomes or artificial viruses • Composed of reconstituted viral proteins structurally [36]
Stealth liposomes • Carrier surface covered with hydrophilic chains [PEG]
• Bypasses phagocytosis and aids in longer circulation in the blood [37, 38]
Archaeosomes • Composed of one or more ether lipids [polar] isolated from Archaebacteria [39]
• These are highly thermostable and resistant to oxidation, chemical and enzymatic hydrolysis. They are highly
resistant to oxidation, enzymatic and chemical hydrolysis and thermostable [40, 41]
Cochleates • These are cigar-shaped multi-layered structure, which consist of negatively charged lipid molecule such as
phosphatidylserine and a divalent cation. Cochleates can deliver positively or negatively charged molecules,
which can be hydrophobic or amphiphilic. Opted for systemic and oral delivery of sensitive moieties espe-
cially antioxidants [42, 43]
Vesicular phospholipid gels • These are highly concentrated dispersions of phospholipids and are of semisolid consistency with vesicles
• Used as parenteral depot formulations for drugs with poor storage stabilities and more leakage rates [44]
Nano liposomes • Longer circulation time in the blood stream and easy penetration into tissues with sustaining effect for days
• Employed for encapsulation and delivery of both vitamin E and ascorbic acid at the oxidation site in the food
system [45]

Fig. 2  Schematic representation of Bangham method, adapted from [49]

• Simple process 5.1.2 Ethanol/Ether Injection Methods

• Straightforward approach
• Used for all kinds of lipid mixtures Batzri and Korninitially described the ethanol injection
 Disadvantages: method in 1973 [50]. In both ethanol and ether injection
• Water soluble drugs exhibit low entrapment efficiency. methods, lipids should be dissolved in an organic solvent
• Difficulty in scaling up. (diethyl ether or ether-methanol mixture or ethanol), fol-
• Removal of organic solvent is troublesome. lowed by injection of mixed solution into aqueous phase,
• Large vesicles without particle size control. where the material should be encapsulated at 55–65ºC or
• Time-consuming method. under reduced pressure to attain liposomes. Further, as ether
• Sterilization is needed. is immiscible with aqueous media, heating is necessary to

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evaporate organic solvent from the prepared liposomes [51]. 5.2.1 Detergent Depletion Method
Hauschild et al. developed the inkjet method as the modern
method of ethanol injection; in this method the drug solu- This technique is used to manufacture homogeneous
tion that is hydrophilic/lipophilic or both was dissolved in liposomes for various drug delivery applications. This
ethanol and transformed to inkjet device for the preparation method determines the solubility of lipid by addition of
of liposomes with tremendous control on particle size and proper detergent in an organic solvent at their critical
for large-scale production [52]. micelle concentration to produce detergent lipid micelles.
Advantages Once after the detergent is removed, the micelles become
increasingly better-off in phospholipid and finally combine
• Ethanol injection method is simple, rapid, reproducible to form liposomes [55]. Detergents should be removed by
and ready to use. dialysis. A commercial device called LipoPrep is used
• Ether injection method forms a concentrated liposome for the elimination of detergents. The dialysis was per-
product with greater entrapment efficiency. formed in dialysis bags placed in large detergent-free
buffers (equilibrium dialysis) [56]. Size and uniformity
Disadvantage of liposomes by detergent method depend on the rate and
extent of detergent removal and on phospholipid to deter-
• Improper mixing may produce heterogeneous liposomes. gent ratio.
• Removal of ethanol is difficult because it forms azeotrope Advantages
with water.
• Various biologically active macromolecules are inactive • Good particle size control
in presence of even low amounts of ethanol. • Simple process
• Possible nozzle blockage (ether system). • Homogenous product

Disadvantages
5.2 Reverse Phase Evaporation Method
• Low concentration of liposomes in the ultimate solu-
Szoka and Papahadjopoulos initially described the reverse tion
phase evaporation method [53], where lipids are dissolved • Low entrapment of lipophilic drug
in an organic solvent and desired drugs are dissolved in an • Time consuming process
aqueous media; further the mixture is sonicated to produce • Removes minute water soluble compounds during
w/o emulsion or inverted micelles, followed by slow removal removal of detergent
of organic solvent using rotary evaporator, leading to con-
version of these micelles into viscous state or gel product.
In this process, at a critical point, the gel state collapses 5.2.2 Microfluidic Channel Method
and some inverted micelles were distributed. The excess
phospholipids form the bilayer around the residual micelles Microfluidic method was established by Jahn et al. [57], for
which results in the formation of liposomes. Modified controlled liposome preparation. In this method, lipids are
reverse phase evaporation method was presented by Handa dissolved in isopropyl alcohol, which then passes through
et al., and the main advantage of this method is the liposome the centre of the dual channels containing aqueous media.
which had high encapsulation efficacy (about 80%) [54]. The stream of the lipids in isopropyl alcohol was subse-
Advantages quently mixing forming liposomes. Size and size distribu-
tion of liposomes are controlled by laminar flow and lipid
• Simple process concentrations in microfluidic channels. This is an effective
• Suitable encapsulation efficacy method to encapsulate the drug directly to attain self-assem-
• Used to encapsulate small, large and macromolecules bled liposomes [58].
Advantages
Disadvantages
• Simple method
• Use of large quantity of organic solvent. • Good particle size control
• Unfit to carry and deliver fragile molecules such as pep- • Low-cost method
tides.  Disadvantages
• Time-consuming method. • Difficulty in removal of organic solvent
• Sterilization is required. • Unsuitable for bulk production

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5.2.3 Heating Method • Not suitable for large-scale processing

• Possibility of clogging the pores
Mortazavi and Mozafari developed the heating method to • Limitation in large-scale production
acquire liposomes [59]. In this method, the phospholipids • Membrane/filter fragility
are hydrated with the help of glycerol, PEG/ethylene glycol • Slow flow through the membrane/filter
with continuous stirring for 1 h at 60° or 120 °C. After cool-
ing, mixture must be centrifuged at 4000 rpm for 15 min to 5.2.5 High Shear Homogenization
obtain the liposomes. Liposomes produced by this method
do not suffer from deterioration of phospholipids, and steri- Homogenization is widely used for the liposomes size reduc-
lization is not mandatory as this process involves high tem- tion and lamellarity. During homogenization the liposome
perature, i.e. 120 °C [60]. Additionally, Mozafari method suspension is continuously pumped through an orifice, and
is the upgraded method of heating method, in which the at very high pressure, it collides with a stainless steel wall
lipid components are hydrated in aqueous media followed in the homogenizer system to produce less size liposomes.
by heating of components without using of organic solvents; Advantages
here stability is increased and used for the delivery of fla-
vourzyme [de-bittering agent] [61]. • Suitable for large-scale production
Advantage • Good particles size control

• Simple and fast process. Disadvantages

• Non-contamination (e.g. organic solvent).
• Sterilization is not needed. • Liposome size distribution is quite broad and variable.
 Disadvantages • Possible metal and oil contamination.
• Low encapsulation efficacy • Use of very high pressure.
• Requires high temperature
• Possible degradation of phospholipids and drugs 5.2.6 Sonication

Sonication method is widely used for the preparation of

5.2.4 Membrane Extrusion Method SUVs. There are 2 sonication techniques used to sonicate
MLVs, namely, bath type sonicator or a probe sonicator
This method is widely used for the conversion of MLVs into under substantial atmosphere [15].
SUVs and LUVs. The liposomes size should be decreased
by passing them through the polycarbonate membrane filter a) Probe sonication: The sonicator tip is directly dipped
having specific pore size at low pressure (< 100 psi). Before into the liposome dispersion. The energy input into lipid
extrusion, LMV are disrupted by freeze–thaw cycles/pre- dispersion is very high, and the coupling energy at the
filtering through large pore size 0.2–1 µm. tip leads to heating. Thus, the vessel should be placed
In this process, the vesicle contents are extruded sev- into a water/ice bath throughout the sonication process
eral times with the dispersion medium during the breaking up to 1 h. More than 5% of lipids can be de-esterified
and resealing of phospholipids layers as they pass through and titanium may pollute the solution.
the polycarbonate membrane. The mean size of vesicles b) Bath sonication: Liposome dispersion is taken in a
obtained by extrusion decreases with increase in the trans- cylinder and is placed into a bath sonicator. In this the
membrane pressure as well as the number of extrusion temperature should be controlled in contrast to probe
cycles. The formed liposomes are called as large unilamel- sonication. The material being sonicated can be pro-
lar vesicles by extrusion having particle size of 120–140 nm tected in a sterile vessel under inert atmosphere.
[62].
Advantages Advantages

• Simple and fast process • Simple process.

• Good particle size control • Particle size is controlled.
• Non-contamination (e.g. organic solvent)
• Several membrane/filter pore sizes available for produc- Disadvantages
ing liposomes
• Very low encapsulation efficacy/internal volume
Disadvantages • Possible degradation of phospholipids and compounds

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• Elimination of large molecules at ­450C, and lyo-protectant dissolves in water at ­450C. Both
• Metal pollution from probe sonicator of the obtained solutions are mixed to form third identi-
• Presence of MLVs along with SUVs cal monophasic solution, followed by filtration, and freeze
drying of solution is performed to produce proliposomes.
Conventional methods used in the preparation of Freeze drying involves 2 steps: in first, the sample is frozen
liposomes have several limitations like low mono-dispers- at − 40ºC and then dried at room temperature which upon
ibility, reduced stability, high organic solvent residue and hydration eventually forms liposomes with mean dimension
toxic effects. Additionally, the organic solvents used in tra- of 100–300 nm [76].
ditional method affect the environment, produce toxicity for Advantages
human health and often degrade the drug. The use of organic
solvents/detergents in preparation of liposomes may lead to • Single step method
protein/drug denaturation and affects membrane property • Used for commercial scale production
[2]. • Increases the shelf-life of the liposomes
• Prevents the physical degradation of liposomes during
5.3 Novel Methods storage

To overcome the stability and formulation issues with con- Disadvantages

ventional methods, numerous innovations in methodology
have taken place, and they are described in further sections, • It is a time and energy consuming process
and liposomes prepared by using various novel techniques • May induce physical alterations, i.e. alterations in vesicle
are presented in Table 3. size
• Loss of encapsulated material can occur
5.3.1 Freeze Drying Method
5.3.2 Dual Asymmetric Centrifugation Method (DAC)
Liu et al. discovered lyophilization monophasic solution
technique for the preparation of liposomes. This method DAC is a unique centrifugation method in which sealed vials
involves dissolving of lipid and drug in tert-butyl alcohol are rotated on the main rotational axis with a definite speed

Table 3  Novel preparation methods with examples

Method Type of vesicles Particle size Drug type EE Example Reference

Reverse phase evaporation MLVs > 500 nm Hydrophilic 30–50% Spirulina LEB-18 [63]
LUVs
Ethanol/ether injection SUVs, SMVs > 100 nm Lipophilic and hydrophilic 99% Beclomethasone dipropionate [64]
MLVs
Heating method GUVs > 1000 nm Lipophilic 30–49% Flavourzyme [62]
Thin film hydration GUVs > 1000 nm Lipophilic 67% Dexamethasone [65]
Freeze drying SUVs < 200 nm Lipophilic 87–93% Calcein, flubiprofen [66]
Amphotericin B
Membrane contractor SUVs, LUVs 100 nm Lipophilic 71% Spironolactone [67]
Dual asymmetric centrifuga- LUVs 70–120 nm Hydrophilic 80% SiRNA-Liposomes [68]
tion
Super critical fluid technology
• SAS 100% Vitamin D3 liposomes(VDL) [69]
• RESS 78.38% Vitamin C [70]
• DELOS 52.2% Anthocyanin [71]
• Super Lip 98% Theophylline [72]
• SCRPE MLVs 100–200 nm Hydrophilic and lipophilic 40% D-( +) Glucose [73]
Lavandin essential oil
• PGSS 6–14% L-α-DPPC and chitosan/ [74]
D-( +)
• ISCRPE (improved 17% GLUCOSE [75]
SCRPE)

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and distance as well as it rotates on its own axis, while in conventional method. The upgraded method of SAS is called
general centrifugation process, the vials should be rotated on as “Continuous Anti-Solvent (CAS)” method.
its own centre axis. The main rotation pushes the sample into
outward direction and rotation around the own centre pushes CAS involves two procedures: CAS1 is a single exit
the sample material into opposite direction due to adhesion process, and in this, liposome solution was injected into an
between the sample and rotating vial. Thus, energy reaches autoclave, and then C­ O2 was filled and organic solution was
into the sample preparation by mechanical turbulence and sprayed under continuous stirring of liposomal solution. The
capitations to produce nano-liposomes with a size of about ­CO2 and liposome suspension was recovered from the same
60 nm for ideal size distribution [66]. valve. CAS2 is a two exit process; in this liposomal suspen-
Advantages sion is extracted from bottom of the autoclave and C­ O2 from
top of the autoclave [81].
• Equipment is small in size and easy to operate. Advantages
• Reproducibility.
• Produce liposomes with small particle size. • Contains low solvent residue.
• Water-soluble drugs have high entrapment efficiency. • Used for the drugs with low solubility.
• No organic solvents are used. • Efficient and environmentally friendly process.
• Particle size is controlled and repeatable.
Disadvantages
Disadvantages
• Needs high amount of phospholipids to obtain sufficient
viscosity. • Causes agglomeration and aggregation of particles.
• Batch scale production. • Presence of residual toxic solvents in the final product.

5.3.3 Supercritical Fluid Methods (SCF) Supercritical ­CO 2 Reverse Phase Evaporation Process
[SCRPE] The SCRPE method was developed by Otake et al.
To overcome the limitations of toxicity and degradability [82]. In SCRPE method, the lipid, organic co-solvent and
of conventional methods, SCF methods are developed as compressed gas are combined in a stirred variable volume
green techniques which involves supercritical anti-solvent cell at a temperature above the lipid phase transition tem-
method (SAS), supercritical reverse phase evaporation perature at 60ºC and 10–30 bar. An aqueous solution is then
method (SCRPE), depressurization of an expanded liquid slowly introduced to the cell, and the pressure is reduced by
organic solution-suspension method (DELOS), supercritical the release of the compressed gas to attain liposomes with
assisted atomization, supercritical assisted liposome forma- the mean diameter of 200–1200 nm. With the decreasing
tion (super Lip) [68], supercritical fluid extraction and par- lipid concentration, the mean size decreases to 100–250 nm.
ticles from gas saturated solutions as a popular alternative The principle of the SCRPE method is similar to the decom-
methods for conventional methods of liposomal prepara- pression method. Otake et al. recently developed a new
tions [77, 78]. ­CO2 is the commonly used supercritical gas, method known as the improved supercritical reverse phase
because it is inflammable, cheap, non-corrosive, non-toxic, evaporation [ISCRPE] technique in which phospholipid and
environmental friendly and used for thermolabile substances drug were sealed in view cell and then temperature raised
[79]. to 60℃, and ­CO2 was passed for 40 min, and the pressure is
released to get liposome dispersion [83].
Supercritical Anti‑solvent [SAS] Method It is widely used Advantages
method for the preparation of proliposomes. SAS is the best
method as it offers simple approach, contains low solvent • Free from organic solvents.
residue and is used for the drugs with low solubility in the • Can encapsulate both water soluble and oil soluble mate-
SCFs (antisolvent) [80]. In this method, supercritical fluid rials.
is passed from the top through the high pressure cham- • Used for commercial scale production.
ber, and then the drug solution is sprayed on the SCF as • Less time-consuming.
small droplets by using atomized nozzle. The liposomes are
attained when hydrated with aqueous solution. The super- Disadvantages
critical ­CO2 acts as antisolvent to the solute but it is miscible
with organic solvent. The liposomes obtained are free from • Low encapsulation efficiency
organic solvent when compared to liposomes produced by • Low liposome stability

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Rapid Expansion of Supercritical Solution [RESS] with baffles and kept at high pressure; further thin bands
Method RESS process is advantageous to achieve microni- are heated thermally to produce SCF. Then the mixture was
zation of poorly water-soluble drug. The important param- passed through the high-pressure formation vessel, and then
eters to be considered in RESS are drug solubility in SCF as aqueous solution containing drug was atomized. The work-
it effects the super saturation and particle size distribution. ing temperature of the saturator and the formation vessel
This process is carried out in two steps: (a) dissolving the was set to 40 °C and pressure at 100 bar. From the bottom of
solid substances in the supercritical fluid and (b) formation the vessel, the liposome suspension was collected, and C ­ O2
of particles by super saturation. Initially, supercritical fluid and ethanol were separated by using a stainless steel separa-
is passed through the extractor at required temperature and tor which is maintained at 30 °C and 10 bar pressure. The
pressure. Then SCF penetrates and dissolves the solid sub- encapsulation efficiency is dependent on the flow rate of the
stances in the extractor at low-pressure chamber, where the aqueous solution, and increased flow rate leads to decreased
solution is depressurized by using a heated nozzle. Addition- entrapment efficiency [84, 85].
ally, cooling by this RESS method may involve nozzle block Advantages
and cause particle de-aggregation [75].
Advantages • Process is continuous and replicable.
• Used to encapsulate hydrophilic drugs.
• Simple and effective process • High entrapment efficiency.
• Reduces the use of organic solvents and can be reused • Low solvent residue.

Disadvantages Disadvantages

• Poor solubility of the polymeric materials in supercritical • Time-consuming process

­ O 2.
C • Requires high pressure
• Huge volume of fluid is needed.
• Expensive.
• May involve nozzle block.
6 Types of Drug Loading in Liposomes
Depressurization of an Expanded Liquid Organic Solution
Main problem associated with liposomes for drug delivery
Suspension Method [DELOS] In this method, the phospho-
applications is drug loading (hydrophilic/hydrophobic).
lipids are dissolved in organic solvent at required tempera-
Most importantly, in a multistep preparation, it is very
ture and pressure, and then the solution is added and mixed
important to distinguish when the drug is to be incorpo-
with the supercritical ­CO2 in a vessel which acts as a co-
rated and at what stage into the system. There are two types
solvent. Further, this solution is depressurized by using a
of drug loading for drug delivery through liposomes, i.e.
nozzle to produce liposomes. The advantage of this method
passive loading and active loading technique.
when compared to PGSS method is thermo-sensitive mate-
Passive loading technique involves the lipid films placed
rials can be used to prepare liposomes, as this process does
on a substrate followed by hydration to form liposomes. In
not require high temperature and this process is carried out
active loading technique, the drug is incorporated after the
under slight working pressure [i.e. 10 MPa at 35 ◦ C] [70].
liposome preparation. Mostly, they are the gradient loading
Advantages
techniques that use buffer ammonium sulphate as gradients.
For active loading of weak base (e.g. ammonium sulphate)
• Simplicity in methodology
into liposomes, trans-membrane gradient is used as driving
• Works under slight conditions
force. For proper loading of weak base with pKa, less than
• Used for thermo-sensitive materials
11 and log P values from − 2.5 to 2 can be used [73].
Disadvantages

• Presence of residual organic solvent 7 Characterization of Liposomes

• Nozzle blockage
7.1 Size and Size Distribution
Super‑Critical Assisted Liposome Formation [Super Lip] For
liposome formation through super lip, as a prime step, the The vesicle size is crucial to determine the in vivo release
lipid dissolves in ethanol and then mixed with pure ­CO2 in of drug-loaded liposomes. The average size of liposomes
a saturator to get an expanded fluid. The saturator is packed depends on the method of preparation and phospholipid

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composition. Various methods are used to evaluate the size phospholipids in liposome influences overall surface charge
and size distribution such as: and blood circulation time of liposomes [89].

(1) Microscopic techniques such as optical microscopy, 7.4 Encapsulation Efficiency/Entrapment Efficiency

scanning electron microscopy (SEM), negative stain TEM
and freeze-fracture TEM. SEM and TEM techniques are Encapsulation efficiency is defined as the percentage
used for imaging of liposomes and also provide informa- of aqueous phase and percentage of water soluble drug
tion about bilayer thickness and inter-bilayer distance of entrapped during preparation of liposomes. It should be
liposome [74]. One of the newly established microscopic expressed as % entrapment/mg of lipid. Enhancing entrap-
methods is atomic force microscopy (AFM), which is a ment efficiency will increase the drug bioavailability [90].
very high-resolution scanning probe microscopy that pro- Various methods such as solid phase extraction, size exclu-
duce 3D micrographs through resolution of nano-meter sion chromatography, hollow fibre-centrifugal ultrafiltration
and ­A0 scale to evaluate the liposome morphology, stabil- and centrifugation ultrafiltration, mini-column centrifuga-
ity, size and dynamic process of lipid nano-capsules [86]. tion and protamine aggregation methods are used to meas-
(2) Hydrodynamic techniques such as ultracentrifugation, ure the entrapment efficiency. Mini-column centrifugation
field flow fractionation and gel exclusion chromatogra- method is used for purification and separation of liposomes,
phy and analytical centrifugation procedures are used to and protamine aggregation method is used for neutral and
estimate molecular mass of compound and also used for negatively charged liposomes.
comparison of size distribution, elution characteristics Indirect method used to measure the encapsulation effi-
and uniformity of the liposomes [87]. ciency is the amount of un-encapsulated drug value that
(3) Diffraction light scattering techniques such as laser should be deducted from the total quantity of the drug to
light scattering, quasi-elastic light scattering and photon be used [91].
correlation spectroscopy give information about the size
of the lipid vesicles. 7.5 In vitro Drug Release Studies

The mean diameter of < 1 µm liposomes can be esti- The in vitro drug release studies are assessed at ­370C
mated by using these techniques. The liposomes size must through in vitro diffusion cell or by using dialysis bag. The
be monitored for different liposome preparations based on cell or bag must be wet through receptor medium contain-
use for parenteral applications, topical use and inhalation ing pH 7.4 buffer with constant stirring under sink condi-
purpose. For controlling of the liposomal size, numerous tions, which mimics the in vivo conditions. At regular time
procedures such as sonication, extrusion and homogeniza- intervals, the required volume of the medium was collected,
tion are available. and the concentration was determined by using HPLC and
UV–Visible spectrophotometry, and at the same time equal
volume of fresh medium was added to receptor media. The
7.2 Lamellarity Determination
cholesterol concentration in liposomal formulations will
affect the release of water soluble drugs, and as the concen-
Lamellarity is defined as the number of lipid bilayers pre-
tration of cholesterol increases, it enhances the release rate
sent around the lipid vesicles. Liposomal lamellarity can be
of the drug [92].
measured by using cryo-electron microscopy, 31P-nuclear
magnetic resonance (NMR) and small angle X-ray scatter-
7.6 In vivo Performance
ing (SAXS) technique that provide information about size,
homogeneity and lamellarity of liposomes [88].
The in vivo behaviour of drug-loaded liposomes might be
influenced by several pharmacokinetic properties of the
7.3 Zeta Potential vesicle. To study the in vivo performance of liposomal drug
delivery systems, the liposomes are administered intrave-
The zeta potential is the key factor that affects the cellu- nously to reveal rapid clearance from spleen and liver. The
lar uptake and targeted drug delivery. The laser Doppler large liposomes having particle size greater than 0.5 µm
electrophoresis and Zetasizer are used to measure the zeta diameter taken by phagocytosis and for liposomes having
potential of the liposomal dispersion by applying an electric particle size less than 0.1 µm were taken up by liver paren-
field based on the scattering of incident laser on the moving chymal cells. Cholesterol incorporated in liposomes will
particles. There are several factors which influence the zeta increase stability by evading the leakage of drug.
potential such as pH, ionic strength and particle concentra- Recently imatinib (anticancer drug)-loaded magnetic
tion. Lipid composition, i.e. positive- and negative-charged liposome nano-composites were synthesized, which was

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functionalized with hyaluronic acid. In vivo behaviour of 9 Applications of Liposomes

this magnetic liposomes is evaluated by tail vein injection
in a mice model, and the fluorescent images were taken 9.1 In Ophthalmic Disorders
at 2, 4, and 8 h after injection. The uptake/retention of
magnetic liposomes takes place at maximum fluorescence Various drugs are used to treat eye disorders, i.e. dry eyes,
signal after 4 h [93]. keratitis and corneal transplant rejection, which should be
applied topically via ointment, solution and suspension form.
Due to several barriers present in the eye, these preparations
7.7 Stability Studies exhibit poor ocular bioavailability. To avoid this hindrance,
liposome formulations are used. In the treatment of dry eyes,
The stability studies are used to determine the shelf life of the liposome suspension and a spray which deliver the drugs
liposomes that involves physical stability, chemical stabil- into conjunctival sac are investigated and developed.
ity, and biological stability. In physical stability, colour The ciprofloxacin/ciprocin is the most widely used eye
change of liposomes can be observed visually or by using drops which is effective against gram +ve and gram −ve
TEM and AFM. Chemical stability involves hydrolysis, bacteria. Here, on comparison of ciprocin eye drops with
oxidation and drug degradation that can be determined ciprocin liposome preparations in rabbits, the area under
by using HPLC, TLC and HPTLC and thiobarbituric acid the curve values for liposome preparations are high caus-
(TBA) test. ing enhanced ocular bio-availability, residence time and low
dose when compared to ciprofloxacin eye drops [94].

9.2 In Cancer Chemotherapy

8 Commercially Available Liposomal
Formulations There is a vast difference between the nature and function
of cancer cell with the normal one, and the cancer cell has
The commercially available formulations of liposomal enhanced permeation rate effect. The tumour has highly per-
drug delivery systems prepared through different methods meable nature, and so the micro and macromolecules can eas-
used for specific purposes are listed in Table 4. ily enter into various tumour cells. Also, specific bio-markers

Table 4  Commercially available liposome formulations

Disease Drug Route of administration Method of preparation Improved profile

Fungal infection Amphotericin B I.V infusion Conventional method Low toxicity, improved
bilayer stability
Breast neoplasms Doxorubicin I.V injection Stealth liposome technology Tumour targeting, high
stability
Analgesic Morphine sulphate Epidural Depofoam technology Prolonged analgesia with low
adverse effects
Viral vaccines Hepatitis A IM/SC injection Detergent removal technique Inactivation of influenza virus
Cancer therapy Daunorubicin I.V injection Conventional method Synergistic effect, targeted
Cytarabin I.V injection Depofoam technology delivery into the tumour
cells
Asthma Terbutaline sulphate Subcutaneous injection Thin film hydration tech- Maximizing therapeutic effi-
nique cacy, reducing undesirable
side effects
Non metastatic osteosar- Mifamurtide I.V infusion Non-PEGylated liposome High safety and tolerability
coma technology
Keratitis Amphotericin B Ocular Conventional method Effective ocular delivery,
sustained drug release
Photodynamic therapy Verteporfin I.V injection Conventional technique More selective targeted deliv-
ery on CNV
Pseudomonas aeruginosa Fluoroquinolones Nebulized aerosol (pulmo- Reverse phase evaporation High encapsulation efficiency
nary) and inhibitory concentration

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are present on cancer cells (amino peptidase and integrin’s), so 11 Recent Patents on Liposomal
the tumour tissue is specific for targeted drug delivery. Formulations
Various drugs are formulated as liposomes, i.e. metho-
trexate, paclitaxel and docetaxel. The anticancer drug release Anticancer agents such as doxycycline [DOX], paclitaxel,
follows two mechanisms into the tumour site viz pH respon- docetaxel, irinotecan, platinates, vincristine, methotrexate
sive (pH difference between the blood (7.4) and tumour cell and etoposide of different liposomal formulations are pat-
(acidic pH)) and the other is polymer degradation by lysozyme ented in 2019 for their high therapeutic effect on the can-
enzyme. Doxorubicin (drug for breast carcinoma) co-loaded cer cells. For instance, Celsion Corp. (NJ, USA) attained
with umbelliprenin into liposomes increases toxicity to the patent [US10251901B2] on the thermosensitive DOX
tested human breast cancer cell lines and improved uniform liposomes where drug uptake was decreased by the retic-
size and distribution of liposomes at target site [95]. uloendothelial system and increased the drug’s circula-
tion time. CSIR disclosed a patent US10426728B2:M2 on
9.3 In Vaccines cationic liposome-encapsulated camptothecin with DOX,
which have shown enhanced anticancer efficacy through
The liposomes are used as adjuvants in vaccine delivery by improvement in the therapeutic index due to synergistic
modifying the surface with different molecules such as peptide activity of DOX and camptothecin. Tumour-targeted DOX
antigens/virus antigens, to boost the immunity and immunolog- carrier with a heavy chain human ferritin was disclosed
ical responses. For example, the membrane proximal external by Institute of Biophysics, Chinese Academy of Sciences
region is an HIV antigen which is on the surface of virion that (PRC), with a patent ID US10195155B2.
has low immunogenicity and increases immune responses [96]. Irinotecan, a semi-synthetic camptothecin deriva-
tive binds to the DNA topoisomerase-I to inhibit nucleic
9.4 In Gene Delivery acid synthesis in the cell cycle (S-phase). An US pat-
ent US10456360B2 was issued by Ipsen Biopharm Ltd.
Cationic liposomes are most commonly used as gene-carriers. (Slough, UK) as irinotecan in formulation as liposomes
Lofectamine 2000 is a cationic liposome commercially used minimized lysophosphatidylcholine (lyso-PC) formation
for gene transfection. Curcumin loaded with STAT3 si-RNA under storage and prior to patient administration.
liposomes are used to treat skin cancer, prepared by Bangham Paclitaxel and docetaxel are the most effective anticancer
method. Liposomes inhibit the growth of B16F10 melanoma agents used to treat lymphomas and leukaemia, and their
cells when compared to free STAT3 si-RNA and free cur- formulations as liposomes have a vast improved profile. Syn-
cumin. Liposomes also deliver the CRISPR or Cas9 gene to core Biotechnology Co. Ltd. improved the paclitaxel for-
treat the genetic disorders and different types of cancers [97]. mulation by freeze-drying paclitaxel liposome formulation
and showed much higher polydispersity after reconstitution
9.5 Other Miscellaneous Applications which is enclosed with patent no. US10413511B2. Shanghai
Weiye Biomedical Technology Co. Ltd. reported the patent
The X-ray, magnetic resonance imaging (MRI) and near-IR WO2019218857A1 on docetaxel palmitate liposomal formu-
fluorescence spectroscopy are broadly used for diagnostic lation using a chelating agents lecithin and DSPE-PEG2000.
imaging. Super paramagnetic liposomes are very efficient Shilpa Medicare Limited patented [WO2019106511A1] for
as MRI contrast agents. Vitamins such as C and tocopherol the information on comparison of a pharmaceutical liposo-
are entrapped in liposomes to increase the thermal structural mal composition comprised of about 0.8% w/w to about 1%
stability of both vitamins [98]. Carotene was encapsulated in w/w of docetaxel.
pro-liposomes to protect the natural pigment from degradation Some platinates such as cisplatin and oxaliplatin were
[99]. Liposome-entrapped with lemongrass oil (bacteriocin- recently patented as liposomal formulations. Oncology
like substance) shows the antibacterial activity, which avoids venture ApS (Denmark) attained a European patent ID
spoilage of cheese [100]. EP3342879B1 for cisplatin liposomes with a secretory
phospholipase A2 (sPLA2) employed for cancer patients.
Mallinckrodt LLC achieved a US patent (US10383823B2
10 Liposomal Formulations Under Clinical for the zwitterionic liposomes with composite of cispl-
Trials atin, phosphatidylcholine lipid, PEG-lipid and cholesterol.
Liposomal encapsulation with oxaliplatin in an aqueous
Various liposomal formulations for numerous applications dispersion holding an external phase of 2-morpholino
are under investigation and are on different phases of clini- ethane sulfonic acid divulged by University of Tokushima,
cal trials; the ongoing formulations of clinical trials in
recent past are listed in Table 5.

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Table 5  Liposomes under different phases of clinical trials (2020–2021)

Drug Disease Sponsor Phase Status

Doxorubicin Hepatocellular carcinoma Celsion III Completed

Amikacin Nontuberculosis mycobacterial lung Insmed Incorporated III Completed
infection
Bupivacaine Post-operative pain Michael Moncure, MD IV Completed
Liposomal curcumin Drug safety SignPath Pharma I Completed
Rhenium nano-liposomes Malignant glioma Plus Therapeutics I and II Recruiting
Liposomated iron Parentral iron therapy after bariatric Hospital Universatari Vall d’Hebron IV Completed
surgery Research Institute
EphA2-targetting DOPC- Solid tumors M.D. Anderson Cancer Center I Active, not recruiting
Encapsulated siRNA
Cabazitaxel Gastroesophageal Adenocarcinoma Weill Medical College of Cornell II Completed
University
Cyclophosphamide Melanoma Sidney Kimmel Comprehensive I Complete
Cancer Center
Dornase alfa Pulmonary infections Georgetown University IV Completed
Azithromycin (T1225) Eye infections Laboratoires Thea I Completed
Influenza vaccination Influenza Hadassah Medical Organization I and II Completed
Cisplatin liposomal Osteosarcoma Metastatic Insumed Incorporated I and II Completed
Liposomal lactoferrin COVID-19 University of Rome Tor Vergata II Completed
SOC therapy III
LEAF-4L6715 COVID-19, sepsis or other causes Institut de cancerologie Strasbourg I and II Recruiting
acute respiratory distress syndrome Europe
Hydroxychloroquine COVID -19 Taiwan Liposome Company I Completed
Bioarginina C Fatigue syndrome Chronic inflamma- University of Milan Not applicable Recruiting
tion (post COVID)
SpFN COVID-19 vac- SARS-CoV-2 infection U.S. Army Medical Research and I Active, not recruiting
cine, QS21 (ALFQ) Development Command
adjuvant

Taiho Pharmaceutical Co. Ltd. (Japan), with a patent ID 12 Conclusion

US10383822B2.
Vinca alkaloids (vincristine, vinblastine and vinorelbine) Liposome-based drug delivery systems are the versatile
are the antimitotic agents that act by disrupting the micro- nano-drug delivery tools that effectively deliver different
tubule assembly through interaction with tubulin with an drugs to specific target sites. Liposomes are used as carriers
eventual arrest of metaphase. A patent granted recently by to increase therapeutic index of several drugs. Researchers
Arbutus Biopharm with ID EP2922529B1 for vincristine has expanded their research on liposomes due to their unique
encapsulation in liposome for therapeutic applications. The characteristics such as non-toxicity, biodegradability, tar-
US10188728B2 patent issued for Temple University (PA, geted drug delivery, biocompatibility, non-immunogenicity,
USA) for their elaborate work on breast cancer treatment and reduced toxicity by improvization of preparation tech-
through immunoliposome formulation through targeted and niques and drug-loading methods. However, most of them
non-targeted delivery strategy employing vinblastine, beva- are limited to laboratory scale. For economical ease and
cizumab and verapamil with a target on Her-2/neu. stability improvement of liposomes, various processes are
A US patent US10307491B2:2019 was granted for an under development. In liposome preparations, SCF technol-
antibiotic mitoxantrone as liposomal formulation by Univer- ogy draws significant attention due to solvent free and high
sity of Michigan for cancer treatment through mitoxantrone drug encapsulation efficiency of liposomes. Based on the
encapsulation in liposomal formulation with one or more current liposomal research, novel methods with novel appli-
cationic lipids and hyaluronic acid. cations and repurposing of drugs are on boon making the
Some of the other recently patented liposomal formula- efficient targeted delivery with reduced toxicity. This review
tions in past 3 years (2019–2021) are listed in Table 6. highlights the recent developments of liposomal formula-
tions with novel formulation methodologies that are easy
scalable at industrial level with description their pros and

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Table 6  Recent patents on liposome formulations (2019–2021)

Title with inventors Patent ID Innovation for patent
Year

Controlled drug release liposome compositions and methods US11147881 Consisting of at least one liposome, or poly/mono valent
thereof 2021 counterion donor or a pharmaceutically acceptable salt, and
Pei Kan, Yun-Long Tseng, Han-Chun Ou, Chun-yen Lai an amphipathic therapeutic agent. This relates to methods of
inhibiting cancer cell growth with reduced toxicity with the
mentioned composition
Composition comprising an onion extract and liposomes US10967036 Onion extract encapsulated liposomes, for treatment/preventing
Peter Boderke, Martina Heberer, Petra Scheppler 2021 scars
Liposome composition and method for producing same US10898435 Innovative method for long-term stability, and a high release rate
Makoto Ono, Kohei Ono, Takeshi Matsumoto, Mikinaga Mori 2021 of a drug controlled by an inner hyper-osmotic water phase
Liposome Containing Compositions and Their Use in Personal US10702475 A liposomal composition of polyglyceryl-10 diacyl surfactant
Care and Food Products 2020 and a water soluble cation in interior aqueous medium to
Richard Rigg, lmani Rigg retain the liposome on a substrate for many rinse treatments
Tailored liposomes for the treatment of bacterial infections US10744089 Liposomes of defined lipid monolayers/bilayers composition for
Eduard Babiychuk, Annette Draeger 2020 the prevention of bacterial (Staphylococcus aureus or Strepto-
coccus pneumonia) infections
Remote loading of sparingly water-soluble drugs into US10722467 For a scalable process, where lipid composition and remote
liposomes 2020 loading agent are optimized, resulting in high drug-to-lipid
Mark E. Hayes, Charles O. Noble, Francis C. Szoka, Jr ratios and prolonged drug retention for less soluble drugs
(< 2 mg/mL) during administration to a subject
Liposome composition for use in peritoneal dialysis US10596114 Liposomal composition for peritoneal dialysis in patients suf-
Jean-Christophe Leroux, Vincent Forster 2020 fering from toxicopathies and for a method of administrating
therapeutically effective amount into the patient’s peritoneal
space
Methods and devices for liposome preparation by centrifuga- US10556216 Methods that impart centrifugal force to a suspension to allow
tion 2020 liposomes through a porous membrane for production of
Oleg Guryev, Tatyana Chernenko, Marybeth Sharkey liposomes
Formulation comprising liposomes US10765633 A liposomal formulation containing 0.7 to 3.0 mol % of eritoran
Hiroshi Ishihara, Katsura Hata, Hiroki Muto, Geoff Hird 2020 or a pharmaceutically acceptable salt and 0.5 to 3.0 mol % of a
PEGylated phospholipid
Liposomes comprising polymer-conjugated lipids and related US10624851 A method of delivering a nucleic acid when encapsulated in a
uses 2020 liposome
Saul Yedgar A method for performing diagnostic imaging with an encapsu-
lated diagnostic agent in a mixed liposome
Methods for treating pathological mixed liposome is adminis-
tered to a subject
Liposomes with ginsenoside as membrane material and prepa- US10639276 The liposomes have a membrane comprising lipids and a ginse-
rations and use thereof 2020 noside
Chong Li, Yahua Wang, Huaxing Zhan
Bi-directionally crosslinked liposomes and method of making US10842746 Bi-directionally crosslinked liposomes with reactive hydrophilic
same 2020 and hydrophobic groups
Kimberly Kam, Zhan Wang, Stephen Morton
Method for preparing liposome frozen powder capable of US10660853 For method using solvents (ethyl acetate and n-hexane) followed
efficiently retaining its bilayer structure 2020 by preparation of frozen powder by inserting in isopropyl
Pahn Shick Chang, Kyung Min Park, Eun Hye Yang, Ho Sup alcohol, ethanol and/or methanol, followed by lyophilization,
Jung for use in food industry with no toxic chloroform
Composition for preventing or treating ischemic diseases, US10617641 A novel method for treating ischemic diseases with loaded vas-
containing liposomes in which VEGF-derived peptides are 2020 cular endothelial growth factor (VEGF) -derived peptides
supported
Hwan Jeong
Microfluidic liposome synthesis, purification and active drug US10434065 Microfluidic methods and systems for large production of
loading 2019 liposomes
Renee Hood, Donald Lad DeVoe
Combinational liposome compositions for cancer therapy US10213385 Method for delivery of active ingredient to a subject employing
Jun Yang, Stephen H. Wu, Cliff J. Herman 2019 multi-component liposomal systems

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288 BioNanoScience (2022) 12:274–291

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