A PROJECT REPORTS ON

ADVERSE DRUG REACTION

SUBMITTED TO

CHHATTISGARH SWAMI VIVEKANAND TECHNICAL UNIVERSITY , BHILAI

IN PARTIAL FULMENT OF THE REQUIRMENT FOR THE DEGREE OF

BECHELOR OF PHARMACY

COLUMBIA INSTITUTE OF PHARMACY, RAIPUR 493111(C.G)

SESSION (2019-2023)
Course BECHELOR OF PHARMACY
Semester 8th
Registration No. BH6070
University Roll No. 302104119009

ANUJ KUMAR SAHU

Date: Supervised By

Dr. S Prakash Rao

Professor
INDEX
 ABSTRACT :

1. Transdermal delivery systems have gained much interest in

recent years owing to their advantages compared to
conventional oral and parenteral delivery systems. They are
noninvasive and self-administered delivery systems that can
improve patient compliance and provide a controlled release
of the therapeutic agents. The greatest challenge of
transdermal delivery systems is the barrier function of the
skin’s outermost layer. Molecules with molecular weights
greater than 500 Da and ionized compounds generally do not
pass through the skin. Therefore, only a limited number of
drugs are capable of being administered by this route.
Encapsulating the drugs in transfersomes are one of the
potential approaches to overcome this problem. They have a
bilayered structure that facilitates the encapsulation of
lipophilic and hydrophilic, as well as amphiphilic, drug with
higher permeation efficiencies compared to conventional
liposomes. Transfersomes are elastic in nature, which can
deform and squeeze themselves as an intact vesicle through
narrow pores that are significantly smaller than its size. This
review aims to describe the concept of transfersomes, the
mechanism of action, different methods of preparation and
characterization and factors affecting the properties of
transfersomes, along with their recent applications in the
transdermal administration of drugs.

Keywords :
transfersomes, nanoencapsulation, transdermal drug delivery
1. Introduction

An efficacious, successful therapeutic treatment cannot be

achieved in most cases, often due to many reasons, such as the
occurrence of hepatic first-pass metabolism, adverse side effects,
the rejection of invasive treatments and poor patient compliance.
Therefore, several drug delivery systems have been developed and
studied over the past decades to overcome these problems. One
promising approach is the use of transdermal delivery systems, as
they are minimally invasive methods without first-pass effects.
However, the barrier function of the skin that prevents or dampens
the transdermal delivery of therapeutic agents has to be addressed.
Nanoencapsulation using a lipid-based vesicular system such as
liposomes has been used to overcome the aforesaid challenge.
Liposomes facilitate drug transport through the skin by three
possible mechanisms: adsorption to the skin surface with a
subsequent transferring of the drug directly from vesicles to skin,
fusion with the lipid matrix of the stratum corneum, thereby
increasing drug partitioning into the skin, and lipid exchange
between the liposomal membrane and cell membrane, facilitating
the diffusion of the drug across the membrane. However, the
problem with conventional liposomes is that they do not deeply
penetrate into the viable skin and blood circulation. Therefore,
liposomes have been widely used as drug carriers for dermal
delivery and not for transdermal delivery. Conventional liposomes
also have limitations, such as the poor encapsulation efficiency of
hydrophilic drugs, an unstable membrane that results in leaky
behavior and a short half-life. These major obstacles have led to the
discovery and development of other novel vesicles such as
niosomes, sphingosomes, bilosomes, chitosomes, transfersomes,
ethosomes and invasomes. Niosomes were first reported in the
early 1970s. They are composed of nonionic surfactants (i.e., of
alkyl or di-alkyl polyglycerol ether class, alkyl ethers, alkyl esters
or alkyl amides); cholesterol and, sometimes, ionic amphiphiles.
The cholesterol provides rigidity to the vesicular bilayer, whereas
nonionic surfactants increase the size and entrapment efficiency of
niosomes
2. Transfersomes

Transferosomes are vesicular carrier systems that are specially

designed to have at least one inner aqueous compartment that is
enclosed by a lipid bilayer, together with an edge activator.

Figure 1

This aqueous core surrounded by a lipid bilayer makes ultra-

deformable vesicles having both self-optimizing and self-regulating
capabilities. In accordance with that, transferosomes are elastic in
nature and can thereby deform and squeeze themselves as intact
vesicles without a measurable loss through narrow pores or
constrictions of the skin that are significantly smaller than the
vesicle size. In contrast to conventional liposomes, which are
comprised of natural (such as egg phosphatidylcholine—EPC and
soybean phosphatidylcholine—SPC) or synthetic (such as
dimyristoyl phosphatidylcholine—DMPC, dipalmitoyl
phosphatidylcholine—DPPC and dipalmitoyl phosphatidyl glycerol
—DPPG) phospholipids , the modified liposomal vesicular system
(transfersomes) is composed of the phospholipid component and
single-chain surfactant as an edge activator. Edge activators (EAs)
function in an exceptional manner as membrane-destabilizing
factors to increase the deformability of vesicle membranes and,
when combined in a proper ratio with an appropriate lipid, gives
the optimal mixture, enabling the transfersomes to become
deformable, as well as ultra-flexible, which results in a higher
permeation capability. Therefore, transfersomes overcome the
major drawbacks of conventional liposomes and penetrate pores
that are much smaller than their own diameters. Furthermore, the
transfersomes maintained their diameters against fragmentation,
even after penetration through the smaller-sized pores. Due to the
usage of EAs in the transfersomal formulation, it has achieved an
enhanced performance compared to the conventional liposomes.
The EAs used in transfersomal formulations can also facilitate the
solubilization of hydrophobic drugs, thereby increasing the drug
entrapment efficiency of the formulations. Moreover, the EAs have
the potential to solubilize and fluidize the skin lipids, resulting in
skin permeation enhancements. The effect of EAs associated in skin
permeations depends on their types and concentrations.
Surfactants are one of many different compounds that act as edge
activators and penetration enhancers. They are known to be
amphiphilic molecules that consist of a lipophilic alkyl chain that is
connected to a hydrophilic head group. Generally, rather than
cationic surfactants, anionic surfactants are furthermore effective
in enhancing the skin penetration, and the critical micelle
concentration is also lower, whereas nonionic surfactants with an
uncharged polar head group are better-tolerated than cationic and
anionic surfactants. Nonionic surfactants are considered less toxic
and less hemolytic, as well as less irritating to cellular surfaces, and
they tend to maintain a near physiological pH in a solution.
Moreover, they have various functions, including acting as
solubilizers, emulsifiers and strong P-glycoprotein inhibitors,
which is useful for enhancing the drug absorption, as well as for
targeting specific tissues. Transfersomal formulations are highly
utilized in “peripheral drug targeting”, “transdermal immunization”
and well-recognized to be a major system for the “transdermal
delivery” of a vast array of therapeutic agents. According to various
research publications, it is evidently known that transfersomes are
capable of transporting low, as well as high, molecular weight (200
≤ MW ≤ 106) bioactive molecules and hydrophilic and lipophilic
molecules through the skin with a transport efficiency greater than
50%. The certain arrangement of cells in the skin prevents
appreciable molecule exchanges between the skin surface and the
skin depth. Even water can only penetrate through the skin barrier
at the rate of only 0.4 mg/cm2/h. It was found that the
transcutaneous permeation rates measured for the molecules with
molecular weights between 18 Da (water) and 750 Da (drugs)
were between 0.1 g/h/cm2 and 1 µg/h/cm2 at most, and the
transferomes were capable of transporting these drug molecules
through the skin of more than 50%, in comparison with the
unentrapped drug. Moreover, it has been reported that
transfersomes can significantly transfer lipophilic fluorescence
markers through the murine skin of more than 50% compared to
that of liposomes .The presence of lipophilic and hydrophilic
moieties in the vesicular structure result in wide range of solubility
of transfersomes . It has been identified that vesicles with sizes
≥600 nm are unable to penetrate deeper skin layers, whereas ≤300
nm reach deeper into the skin. However, those vesicles with ≤70
nm have exhibited a maximum deposition of the contents in both
the viable epidermal as well as the dermal layers of the skin.
Furthermore, it has been reported that statistically enhanced skin
penetration was exhibited by transfersomes with 120-nm sizes
compared to larger ones. The optimized delivery, improved
bioavailability and promising stability of phytoactives in herbal
formulations are also ensured by these novel transfersome
delivery systems. Therefore, herbal ingredients can be
encapsulated in transfersomes to supply promising skin care and
various therapeutic benefits. It was described in several published
research papers that, due to vast skin-penetration capabilities,
transfersomes are able to create skin drug depots for a sustained
drug release, deliver therapeutic agents into deeper skin layers or
transport drugs into the systemic circulation. Hence, transfersomes
propose a good potential to provide a whole new perspective for
the rational drug delivery.
 Advantages of Transfersomes as Vesicle-based Transdermal
Drug Delivery Systems :

 Transfersomes carriers are composed of hydrophilic and

hydrophobic moieties, which result in becoming a unique drug
carrier system that can deliver therapeutic agents with wide range
of solubility.

 Transfersomes are able to squeeze themselves through

constrictions of the skin barrier that are very narrow, such as 5 to
10 times less than the vesicle diameter, owing to their ultra-
deformability and elastic properties.

 High vesicle deformability facilitates the transport of drugs across

the skin without any measurable loss in intact vesicles and can be
used for both topical, as well as systemic, treatments.

 Transfersomes carriers are very versatile and efficient in

accommodating a variety of agents nearly independent of their
size, structure, molecular weight or polarity.

 They are made up of natural phospholipids and EAs, therefore

promisingly biocompatible and biodegradable.

Limitations of transfersomes :
 Transfersomes are considered as chemically unstable due to their
tendency to oxidative degradation. The oxidation of transfersomes
can be significantly decreased when the aqueous media is degassed
and purged with inert gases, such as nitrogen and argon.

 Storage at a low temperature and protection from light will also

reduce the chance of oxidation. Post-preparation processing, such
as freeze-drying and spray-drying, can improve the storage
stability of transfersomes.

 Another obstacle of utilizing transfersomes as a drug delivery

system is the difficulty to achieve the purity of natural
phospholipids. Therefore, synthetic phospholipids could be used as
alternatives.

 The expensiveness of transfersomal formulations is associated

with the raw materials used in lipid excipients, as well as the
expensive equipment needed to increase manufacturing. Hence,
the widely used lipid component is phosphatidylcholine, because it
is relatively low in cost

3. Mechanism of Action

Vesicles are known as colloidal particles, which are an aqueous

compartment enclosed by a concentric bilayer that are made-up of
amphiphilic molecules. They are very useful as vesicular drug
delivery systems, which transport hydrophilic drugs encapsulated
in the inner aqueous compartment, whereas hydrophobic drugs
are entrapped within the lipid bilayer. With regard to
transfersomes, they are highly deformable (ultra-flexible) and self-
optimizing novel drug carrier vesicles, in which their passage
across the skin is mainly associated with the transfersomes’
membrane flexibility, hydrophilicity and the ability to maintain the
vesicle’s integrity (Figure 2).

Figure 2
The mechanism of action of transfersomes.

They efficiently penetrate through the intact skin if applied under

nonocclusive conditions; this specific nonocclusive state of the skin
is required mainly to initiate a transepidermal osmotic gradient
across the skin. According to the study done by Cevc and Blume,
hydrotaxis (xerophobia) is the permeation mechanism of
transfersomes, which is further described as the transferosome’s
moisture-seeking tendency towards deeper skin layers rather than
dry outer background due to the state of moisture evaporation
from the transfersomal formulation following its application on the
skin (nonocclusive condition). The transdermal water activity
difference, which originates due to the natural transdermal
gradient, creates a significantly strong force that acts upon the skin
through transfersomes vesicles, which enforce the widening of
intercellular junctions with the lowest resistance and thereby
generate transcutaneous channels 20–30 nm in width. These
created channels allow the transfer of ultra-deformable, slimed
transfersomes across the skin with respect to the hydration
gradient. Moreover, the osmotic gradient develops as a result of
evaporation of the skin surface water due to body heat, which
exerts its action as the driving force to facilitate the flexible
transport across the skin to deliver therapeutic agents from the
site of application to the target area for local or systemic
treatments in effective therapeutic concentrations and minimum
systemic toxicity. Transfersomes demonstrate a higher permeation
efficiency (through small skin channels) compared to conventional
liposomes but have a similar bilayered structure that facilitates the
encapsulation of lipophilic and hydrophilic, as well as amphiphilic,
drugs. Transfersomes vary from liposomes, primarily due to their
softer, better adjustable and ultra-deformable artificial
membranes. Interdependency of the local composition, as well as
the shape of the lipid bilayer, makes the vesicles both self-
optimizing and self-regulating. This property enables the
transfersomes vesicles to cross numerous transport barriers
efficiently. Therefore, transfersomes are supramolecular entities
composed of at least one type of amphipathic agent and, by the
addition of at least one type of bilayer-softening agent (edge
activator), result in greatly increased lipid bilayer flexibility and
permeability. Certain transfersomes have some amounts of alcohol
(ethanol or propylene glycol) in their compositions as penetration
enhancers and, also, used as cosolvents that have good solvating
power. Ethanol has been proposed to induce modifications of the
 lipid bilayer polar head region. Following penetration, ethanol
increases the fluidity of the intercellular lipid matrix and later on
results in decreasing the density of the lipid lamellae.
Transfersomes can penetrate through the stratum corneum and
reach the target sites, including the dermis and blood circulation.
Their penetration ability depends on the deformability of the
transfersomal membrane, which can be attributed to the vesicle
compositions. Therefore, the most suitable vesicle compositions
must be identified through conducting individually designed
experimental procedures for each therapeutic agent to obtain the
most appropriate carriers with optimum deformability, drug
carrying capacity and stability.

4. Composition of Transfersomes

Transfersomes are generally composed of

 firstly, the main ingredient, an amphipathic ingredient (e.g., soy

phosphatidylcholine, egg phosphatidylcholine, etc.) that can be a
mixture of lipids, which are the vesicle-forming components that create
the lipid bilayer .
 secondly, 10–25% surfactants/edge activators; the most commonly
used edge activators in transfersome preparations are surfactants as
sodium cholates; sodium deoxycholate; Tweens and Spans (Tween 20,
Tween 60, Tween 80, Span 60, Span 65 and Span 80) and dipotassium
glycyrrhizinate, which are biocompatible bilayer-softening compounds
that increase the vesicles’ bilayer flexibility and improve the
permeability .
 about 3–10% alcohol (ethanol or methanol), as the solvent and, finally,
hydrating medium consist with either water or a saline phosphate
buffer (pH 6.5–7)

In an aqueous environment, the phospholipids self-assemble into

flexible lipid bilayers and close to form vesicles . The biocompatible
membrane softeners, which are also known as edge activators, are
single-chain surfactants that incorporate into the transfersomes
structure and facilitate the destabilization of the vesicle’s lipid bilayer
and enhance its fluidity and elasticity . The total amount of surfactants
and the proper ratios of individual surfactants to phospholipids are
responsible for the control of vesicles’ membrane flexibility and
minimizing the risk towards vesicle ruptures in the skin . This result
promotes transfersomes to follow the natural osmotic gradient across
the epidermis following application under a nonocclusive manner [. In
summary, the penetration-enhancing effect of these vesicles depends
on the concentrations and the types of surfactants, the types of lipids,
the size shape and elasticity of the vesicles.

5. Preparation Methods

Even though there are various patented procedures of transfersome

preparation, there is no general preparation protocol or a specific
formula for this process .

Therefore, the best preparation conditions and vesicles compositions

must be identified, designed and optimized through conducting
individually designed experimental procedures for each therapeutic
agent to obtain the most appropriate carriers with optimum
deformability, drug carrying capacity and stability .

The conventional method of transfersome preparation is the thin film

hydration technique, also known as the rotary evaporation-sonication
method. Other modified preparation methods are vortexing-sonication,
the modified handshaking process, suspension homogenization,
centrifugation process, reverse-phase evaporation method, high-
pressure homogenization technique and ethanol injection method.
Each method is generally described as follows:
5.1. Thin Film Hydration Technique/Rotary
Evaporation-Sonication Method
The phospholipids and edge activator (vesicle-forming ingredients) are
dissolved in a round-bottom flask using a volatile organic solvent
mixture (example: chloroform and methanol in a suitable (v/v) ratio).
The lipophilic drug can be incorporated in this step.
In order to form a thin film, the organic solvent is evaporated above
the lipid transition temperature under reduced pressure using a rotary
vacuum evaporator.
Keep it under vacuum to remove the final traces of the solvent. The
deposited thin film is then hydrated using a buffer solution with the
appropriate pH (example: pH 7.4) by rotation for a respective time at
the corresponding temperature. The hydrophilic drug incorporation
can be done in this stage.
The resulting vesicles are swollen at room temperature and sonicated
in a bath or probe sonicator to obtain small vesicles. The sonicated
vesicles are homogenized by extrusion through a sandwich of 200 nm
to 100 nm polycarbonate membranes .

5.2. Vortexing-Sonication Method

The phospholipids, edge activator and the drug are mixed in a

phosphate buffer. The mixture is then vortexed until a milky
transfersomal suspension is obtained. It is then sonicated, using a bath
sonicator, for a respective time at room temperature and then
extruded through polycarbonate membranes (example: 450 and 220
nm)
5.3. Modified Handshaking Process

The modified handshaking method has the same basic principle as the
rotary evaporation-sonication method. In the modified handshaking
process, the organic solvent, the lipophilic drug, the phospholipids and
edge activator are added in a round-bottom flask. All the excipients
should completely dissolve in the solvent and obtain a clear
transparent solution. Then, the organic solvent is removed by
evaporation while handshaking instead of using the rotary vacuum
evaporator. In the meantime, the round-bottom flask is partially
immersed in the water bath maintained at a high temperature
(example: 40–60 °C). A thin lipid film is then formed inside the flask
wall. The flask is kept overnight for complete evaporation of the
solvent. The formed film is then hydrated with the appropriate buffer
solution with gentle shaking at a temperature above its phase
transition temperature. The hydrophilic drug incorporation can be
done in this stage.

5.4. Suspension Homogenization Method

Transfersomes are prepared by mixing an ethanolic phospholipid

solution with an appropriate amount of edge activator. The prepared
suspension is subsequently mixed with buffer to yield a total lipid
concentration. The resulting formulation is then sonicated, frozen and
thawed respectively two to three times.

5.5. Centrifugation Process

The phospholipids, edge activator and the lipophilic drug are dissolved
in the organic solvent. The solvent is then removed using a rotary
evaporator under reduced pressure at the respective temperature. The
remaining traces of solvent are removed under vacuum. The deposited
lipid film is hydrated with the appropriate buffer solution by
centrifuging at room temperature. The hydrophilic drug incorporation
can be done in this stage. The resulting vesicles are swollen at room
temperature. The obtained multilamellar lipid vesicles are further
sonicated at room temperature .
5.6. Reverse-Phase Evaporation Method
The phospholipids and edge activator are added to a round-bottom
flask and dissolved in the organic solvent mixture (example: diethyl
ether and chloroform). The lipophilic drug can be incorporated in this
step. Then, the solvent is evaporated using rotary evaporator to obtain
the lipid films. The lipid films are redissolved in the organic phase
mostly composed of isopropyl ether and/or diethyl ether.
Subsequently, the aqueous phase is added to the organic phase, leading
to a two-phase system. The hydrophilic drug incorporation can be done
in this stage. This system is then subjected to sonication using a bath
sonicator until a homogeneous w/o (water in oil) emulsion is formed.
The organic solvent is slowly evaporated using rotary evaporator to
form a viscous gel, which then becomes a vesicular suspension .

5.7. High-Pressure Homogenization Technique

The phospholipids, edge activator and the drug are uniformly

dispersed in PBS or distilled water containing alcohol and followed by
ultrasonic shaking and stirred simultaneously. The mixture is then
subjected to intermittent ultrasonic shaking. The resulting mixture is
then homogenized using a high-pressure homogenizer. Finally, the
transfersomes are stored in appropriate conditions.

5.8. Ethanol Injection Method

The organic phase is produced by dissolving the phospholipid, edge

activator and the lipophilic drug in ethanol with magnetic stirring for
the respective time, until a clear solution is obtained. The aqueous
phase is produced by dissolving the water-soluble substances in the
phosphate buffer. The hydrophilic drug incorporation can be done in
this stage. Both solutions are heated up to 45–50 °C. Afterwards, the
ethanolic phospholipid solution is injected dropwise into the aqueous
solution with continuous stirring for the respective time. Ethanol
removal is done by transferring the resultant dispersion into a vacuum
evaporator and then sonicating for particle size reduction.
6. Factors Affecting Properties of Transfersomes
In the process of obtaining an optimized formulation of transfersomes,
there are number of process variables that could affect the properties
of the transfersomes. These variables basically involve the
manufacturing of transfersomal formulations, which are identified as
follows:

6.1. Effect of Phospholipids: Edge Activator Ratio

The phospholipid: Edge activator (lecithin:surfactant) should be an

optimized ratio due to the fact that this greatly affects the entrapment
efficiency, vesicle size and permeation ability. In general, it has been
reported that the EE could be reduced due to the presence of a higher
surfactant concentration. This may be due to the result of increased
vesicles’ membrane permeability because of the arrangement of
surfactant molecules within the vesicular lipid bilayer structure, which
could generate pores within the vesicular membrane and lead to an
increased fluidity and prompt the leakage of the entrapped drug . A
further increase in the edge activator content may lead to pore
formation in the bilayer and a reduced permeation ability of the
vesicles , whereas the incorporation of low concentrations of
surfactants may result in growth of the vesicle size. In addition, the
decrease in vesicles size at high phospholipid concentrations has been
reported in various studies .

6.2. Effect of Various Solvents

Various solvents such as ethanol or methanol are used. Selection of the

appropriate solvent depends on the solubility of all the formulation
ingredients in the solvent and their compatibility with the solvent.
Preferably, all the excipients, including the drug, should completely
dissolve in the solvent and should obtain a clear transparent solution
to produce a better film-forming ability and good stability after
hydration . Solvents used in the formulation can also exert their
function as penetration enhancers that improve drug flux through the
membrane. According to Williams and Barry (2004), ethanol was used
in various studies to enhance the flux of hydrocortisone,
 5-fluorouracil, estradiol and levonorgestrel through rat skin . For an
example, ethanol increases the permeation through different
mechanisms, such as increasing the drug solubility in vesicles by acting
as a solvent, moreover permeating into the stratum corneum and
altering the solubility properties of the respective tissue and,
consequently, improving the drug partitioning into the membrane.
Increasing the ethanol concentration in the formulation may result in a
decrease in the %EE, which could be attributed to the increased
permeability of the vesicular phospholipid bilayer. This may promote
the consequent leakage of the encapsulated drug .

6.3. Effect of Various Edge Activators (Surfactants)

Deformability, as well as the entrapment efficiency of transfersome

vesicles, are affected by the type of edge activators used in their
formulations. This could be due to the difference in the chemical
structure of the EA . Generally, the vesicle size decreases by increasing
the surfactant concentration, the hydrophilicity of the surfactant head
group, carbon chain length and the hydrophilic lipophilic balance
(HLB). The three surfactants, including tween 80, span 80 and sodium
deoxycholate, were used to prepare the transfersomes, and a reduction
of the vesicle size was found when the higher surfactant concentration
used. This might be due to the fact that the high surfactant
concentrations (more than 15%) induce micelle formation rather than
vesicle formation . A small polydispersity index (PDI) was reported
with the higher surfactant concentration. A small PDI is responsible for
consistent size distribution, which is thought to be an important factor
for the reduction of interfacial tension and provides a homogeneous
formulation. Additionally, an increased surfactant concentration may
lead to an increase in charge of the vesicles, which results in a
reduction of vesicle aggregation and enhances the stability of the
system.
 In addition, surfactant properties are one of the properties that are
responsible for the entrapment efficiency of the vesicles, as, for an
example, the entrapment of a lipophilic drug would be enhanced with
the use of a surfactant with a low HLB value. Moreover, it has been
reported that higher surfactant concentrations will increase the
formation of the vesicle number, which leads to a higher volume of the
hydrophobic bilayer domain that is available for the entrapment of
hydrophobic drugs. However, if the amount of lipophilic drug exceeds
the vesicular loading capacity, it may disrupt the vesicular membrane,
leading to drug leakage, lowering the entrapment efficiency and skin
permeation ability . Furthermore, the membrane permeability of
vesicles depends on the carbon chain length and transition
temperature of the surfactant. The optimum amount of surfactant used
in the formulation depends on the packing density of the phospholipid
used and the surfactant-phospholipid interaction . The presence of
surfactants can have an impact on the permeation property of
transfersomes. According to a study by Cipolla et al. (2014), the
amount of drug (ciprofloxacin) released was dependent on the
concentration, as well as the type of surfactant used, and using Tween
80 significantly increased the release .

6.4. Effect of the Hydration Medium

The hydrating medium may consist of either water or saline phosphate

buffer (pH 6.5–7). The pH level of the formulation should be suitable to
achieve a balance between both the formulation properties and
biological applications, as well as the route of administration. The lipid
bilayer of transfersomes mimics the phospholipid layer of the cell
membrane, and only unionized drugs remain membrane-bound to the
phospholipid bilayer and penetrate through the intracellular route . It
is important to use the suitable pH of the hydration medium, which
keeps the drug unionized to increase the entrapment and permeation
of the drug.
7. Characterization of the Transfersomes
There are several published methods used to determine the
characterization parameters of the transfersomes, such as the vesicle
shape and size, size distribution, polydispersity index, zeta potential,
number of vesicles for cubic mm, entrapment efficiency, degree of
deformability and skin permeability measurements , which are
beneficial for the optimization of the transfersomal formulation. Each
characterization method mentioned above is explained in detail below.

7.1. Vesicle Size, Zeta Potential and Morphology

The vesicle size is one of the important parameters during

transfersome preparation, batch-to-batch comparison and scale-up
processes. During storage, the changing of the vesicle size is an
important variable in terms of the physical stability of the formulation.
Vesicles smaller than 40 nm are prone to fusion processes because of
the high curvature state of their bilayer membranes, whereas much
larger and electroneutral transfersomes are aggregated through van
der Waals interactions due to relatively greater membrane contact
areas. Vesicle size is a factor that influences the ability to encapsulate
the drug compounds in transfersomes. For lipophilic and amphiphilic
agents, a high lipid-to-core ratio is favored, while a larger aqueous core
volume is preferred for the encapsulation of hydrophilic compounds.
Generally, the dynamic light scattering (DLS) method or photon
correlation spectroscopy (PCS) can be used to determine the vesicle
diameter. The vesicle’s suspension can be mixed with an appropriate
medium, and the vesicular size measurements can be obtained in
triplicate. Moreover, as another approach, the sample can be prepared
in distilled water and filtered through a 0.2 mm membrane filter. The
filtered sample is then diluted with filtered saline to measure the size
of the vesicles by DLS or PCS. Moreover, the DLS method-associated
computerized inspection system by Malvern Zetasizer can be used for
the determination of the vesicle size and size distribution, whereas the
structural changes are observed by transmission electron microscopy
(TEM). The zeta potential is measured by the electrophoretic mobility
technique using Malvern Zetasizer. The visualization of transfersome
vesicles can be done by using the phase contrast microscopy or TEM .
7.2. Number of Vesicles Per Cubic mm

This parameter is important for the optimization of the composition of

the transfersomes and other process variables [1,3,53]. Unsonicated
transfersomal formulations are diluted five times using 0.9% sodium
chloride. A hemocytometer with an optical microscope is used to study
this sample. The transfersomes with a vesicle size of more than 100 nm
can be observed by optical microscope [88,89]. The number of
transfersomes in small squares are counted and calculated using the
following formula:

Total number of transfersomes per cubic mm =

(Total number of transfersomes counted × dilution factor ×4000)
Total number of squares counted
(1)

7.3. Entrapment Efficiency (%EE)

The entrapment efficiency (%EE) is the amount of drug entrapped in

the formulation. The EE is determined by separating the unentrapped
drug from the vesicles using various techniques, such as mini-column
centrifugation. In this process, direct or indirect methods can be used
to determine the %EE. After ultracentrifugation, the direct approach
would be removing the supernatant followed by disrupting the
sedimented vesicles using a suitable solvent that is capable of lysing
the sediment. Subsequently, the resulting solution can be diluted and
filtered using a syringe filter (0.22–0.45 µm) to remove the impurities.
The drug content is determined by employing analytical methods, such
as modified high-performance liquid chromatography (HPLC) or
spectrophotometrically, which depends on the analytical method of the
active pharmaceutical ingredient (API) . The percentage drug
entrapment (the entrapment efficiency) is expressed as:

%Entrapment efficiency = Amount of the drug entrappedTotal am

ount of the drug added×100
(2)
The indirect approach to determine the %EE is diluting the
supernatant using a suitable solvent and filtering it to remove the
impurities. The concentration of the drug in the supernatant is
determined as the free drug by an appropriate analytical method.
Thereby, the percentage drug entrapment is expressed as:

%Entrapment efficiency = Total amount of the drug added − Amou

nt of the free drugTotal amount of the drug added×100
(3)

7.4. Degree of Deformability

This parameter is important, as it affects the permeation of the

transfersomal formulation. This study is done using pure water as the
standard. The preparation is passed through many microporous filters
of known pore sizes between 50 to 400 nm. The particle size, as well as
the size distribution, are noted after each pass using DLS
measurements. The degree of deformability is expressed as:

D=J(rvrp)
(4)

where D = degree of deformability, J = amount of suspension extruded

during 5 min, rv = size of the vesicle and rp = pore size of the barrier.

7.5. In Vitro Drug Release

The in vitro drug release profile can provide fundamental information

on the formulation design and details on the release mechanism and
kinetics, enabling a scientific approach to optimize the transfersomal
formulation. The in vitro drug release of transfersomes is typically
evaluated in comparison to the free drug or the reference product.
Various research studies have evidently provided successful data
related to the drug release profiles of developed transfersome
formulations. Celecoxib transfersomal gel for the rheumatoid arthritis
treatment showed the release of 75% of the entrapped drug within 6 h,
which is more than a 30% increment relative to the commercial gel .
Ketoconazole-loaded transfersomal gel showed an initial burst of the
drug release of 40.67%, which was higher than that of ketoconazole
suspension (27.35%) after 6 h. The in vitro release profile of lidocaine
from the transfersomal vesicles showed more than 80% of the drug
released after 6 h. In brief, Franz diffusion cells are employed in the in
vitro drug release study. The donor chamber is fixed to the receptor
chamber by means of adhesive tape. The fluid in the receptor chamber
is constantly stirred by a magnetic bar. As normal skin surface
temperature is approximately 32 °C , therefore, in the release study,
the temperature of the receptor fluid should be kept at the in vivo skin
surface temperature of 32 ± 1 °C [38,96]. A mixed cellulose ester
membrane of an average pore size of 0.45 µm is used. The membranes
are soaked in the release media (phosphate buffer) at room
temperature overnight in order to allow the membrane pores to swell.
The aliquots of 1 mL of the receptor medium are withdrawn at
appropriate time intervals (such as 0, 0.5, 1, 2, 3, 4, 5 and 6 h), and
simultaneously, the receptor medium is replaced by an equal volume of
the fresh PBS to maintain the sink conditions. The obtained samples
can be analyzed by using appropriate methods such as UV, HPLC and
high-performance thin layer chromatography (HPTLC)

7.6. In Vitro Skin Permeation Studies

This study is performed to determine the transport efficiencies of the

transdermal delivery systems and identify the factors that increase the
transdermal flux of the drugs, which is typically expressed in units of
μg/cm2/h. The information obtained from this study can also be used
to predict in vivo behaviors from different transdermal delivery
systems and used for the optimization of the formulation prior to
performing more expensive in-vivo studies. Ideally, the human skin
should be used for the evaluation of permeation properties of
candidate formulations. However, the limited availability, ethical
problems and religious restrictions of the human skin make it less
attractive for the permeation study. Various animal models, such as
primate, porcine, rat, mouse, guinea pig and snake skins, have been
suggested as more accessible substitutes for human skin. However, it
should be noted that percutaneous absorption through various animal
skins may differ significantly from the results obtained with human
skin models. According to the published data, it is evidently suggested
that pig skin is the most suitable animal model for human skin due to
the fact that the fluxes through the skin, as well as the concentrations
in the skin, were exhibited to be of the same order of magnitude for
both of those tissues, with minor differences of, at most, two or four-
fold, respectively.

7.7. Stability of Transfersomes

The stability of transfersome vesicles can be determined by assessing

the structure and the size of vesicles with respect to time. DLS and TEM
can be used for the determination of the mean size and structural
changes, respectively. The optimized transfersomal formulations can
be stored in tightly sealed amber vials at different temperature
conditions. According to ICH (International Conference on
Harmonization) guidelines, under the stability testing of new drug
substances and products, the general case for the storage condition is
described as, for the long term, 25 ± 2 °C/60% relative humidity (RH) ±
5% RH or 30 ± 2 °C/65% RH ± 5% for 12 months and, for accelerated
testing, 40 ± 2 °C/75% RH ± 5% for six months. Drug products
intended for refrigeration should be subjected to long-term storage at a
condition of 5 ± 3 °C for 12 months and accelerated study for 25 ± 2
°C/60% RH ± 5% RH for six months. A significant change for the drug
product is defined as the failure to meet its specifications.

8. Applications of Transfersomes as the Transdermal

Delivery System

Over the past decades, the applications of the transfersomes in the field
of transdermal drug administration have been extensively studied.
Some of these applications are described in the section below.

8.1. Delivery of Antioxidants

In 2017, Avadhani et al. developed nanotransfersomes containing

epigallocatechin-3-gallate (EGCG) and hyaluronic acid by using a
modified thin-film hydration method followed by the high-pressure
homogenization technique in order to enhance their efficacies as UV
radiation protectors, antioxidants and antiaging substances [100]. In
2019, Wu et al. prepared transfersomes combined with resveratrol
using the high-pressure homogenization technique.
8.2. Delivery of Anticancer Drugs

A research conducted by Jiang et al. in 2018 was associated with the

topical chemotherapy of melanoma by transfersome-embedded
oligopeptide hydrogels containing paclitaxel prepared by the thin-film
dispersion method. Transfersomes composed of phosphatidylcholine,
tween80 and sodium deoxycholate were shown to effectively penetrate
into tumor tissues.

8.3. Delivery of Corticosteroids

The biological activity and characteristics of halogenated corticosteroid

triamcinolone-acetonide-loaded transfersomes prepared by the
conventional thin-film hydration technique were studied by Cevc and
Blume in 2003 and 2004. The results showed that transfersomes had
increased the biological potency and prolonged effect, as well as the
reduced therapeutic dosage.

8.4. Delivery of Anti-Inflammatory Drugs

Diclofenac sodium, celecoxib, mefenamic acid and curcumin-loaded

transfersomes were developed and studied for the purpose of topical
administration by several research groups. Research findings
suggested that transferomes could improve the stability and efficacy of
the anti-inflammatory drugs

9. Conclusions

Transfersomes are ultra-deformable carriers that facilitate the delivery

of a diverse array of drug molecules across the skin barrier with
superior efficacy compared to the conventional vesicular systems. The
osmotic gradient is the main driving force for the transport of
transfersomes into the deeper skin layers. Importantly, transferosomes
are specifically designed vesicular systems that need to be optimized in
accordance with individual cases of drugs of interest to achieve the
most effective formulations and ultimate pharmacological responses.
Further scientific studies associated with transfersomes may lead to
novel promising therapeutic approaches against many types of
diseases.
Acknowledgments

The authors acknowledge Department of Pharmaceutics and Industrial

Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn
University and Pharmaceutical Sciences and Technology Graduate
Program for administrative support.
References
1. Chaurasiya P., Ganju E., Upmanyu N., Ray S.K., Jain P. Transfersomes: A novel technique for
transdermal drug delivery. J. Drug Deliv. Ther. 2019;9:279–285.
doi: 10.22270/jddt.v9i1.2198. [CrossRef] [Google Scholar]

2. Modi C., Bharadia P. Transfersomes: New dominants for transdermal drug delivery. Am. J.
PharmTech. Res. 2012;2:71–91. [Google Scholar]

3. Jain A.K., Kumar F. Transfersomes: Ultradeformable vesicles for transdermal drug

delivery. Asian J. Biomater. Res. 2017;3:1–13. [Google Scholar]

4. Elsayed M.A., Abdallah O.Y., Naggar V.F., Khalafallah N.M. Lipid vesicles for skin delivery of
drugs: Reviewing three decades of research. Int. J. Pharm. 2007;332:1–16.
doi: 10.1016/j.ijpharm.2006.12.005. [PubMed] [CrossRef] [Google Scholar]

5. Touitou E., Junginger H.E., Weiner N.D., Nagai T., Mezei M. Liposomes as carriers for topical
and transdermal delivery. J. Pharm. Sci. 1994;83:1189–1203.
doi: 10.1002/jps.2600830902. [PubMed] [CrossRef] [Google Scholar]

6. Yadav D., Sandeep K., Pandey D., Dutta R.K. Liposomes for drug delivery. J. Biotechnol.
Biomater. 2017;7:1–8. doi: 10.4172/2155-952X.1000276. [CrossRef] [Google Scholar]

7. Cevc G. Transfersomes, liposomes and other lipid suspensions on the skin: Permeation
enhancement, vesicle penetration, and transdermal drug delivery. Crit. Rev. Ther. Drug Carr.
Syst. 1996;13:257–388. doi: 10.1615/CritRevTherDrugCarrierSyst.v13.i3-4.30. [PubMed]
[CrossRef] [Google Scholar]

8. Rajan R., Jose S., Mukund V.P.B., Vasudevan D.T. Transferosomes—A vesicular transdermal
delivery system for enhanced drug permeation. J. Adv. Pharm. Technol. Res. 2011;2:138–143.
doi: 10.4103/2231-4040.85524. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

9. Lymberopoulos A., Demopoulou C., Kyriazi M., Katsarou M., Demertzis N., Hatziandoniou S.,
Maswadeh H., Papaioanou G., Demetzos C., Maibach H., et al. Liposome percutaneous
penetration in vivo. Toxicol. Res. Appl. 2017;1:1–6.
doi: 10.1177/2397847317723196. [CrossRef] [Google Scholar]

10. Xu X., Khan M.A., Burgess D.J. Predicting hydrophilic drug encapsulation inside unilamellar
liposomes. Int. J. Pharm. 2012;423:410–418. doi: 10.1016/j.ijpharm.2011.12.019. [PubMed]
[CrossRef] [Google Scholar]

11. Szoka F., Papahadjopoulos D. Procedure for preparation of liposomes with large internal
aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci.
USA. 1978;75:4194–4198. doi: 10.1073/pnas.75.9.4194. [PMC free article] [PubMed]
[CrossRef] [Google Scholar]

12. Crommelin D.J.A., Fransen G.J., Salemink P.J.M. Stability of liposomes on storage. Target.
Drugs Synth. Syst. 1986:277–287. doi: 10.1007/978-1-4684-5185-6_20. [CrossRef] [Google
Scholar]
13. Ge X., Wei M., He S., Yuan W. Advances of non-ionic surfactant vesicles (niosomes) and
their application in drug delivery. Pharmaceutics. 2019;11:55.
doi: 10.3390/pharmaceutics11020055. [PMC free article] [PubMed] [CrossRef] [Google
Scholar]

14. Marianecci C., Di Marzio L., Rinaldi F., Celia C., Paolino D., Alhaique F., Esposito S., Carafa M.
Niosomes from 80s to present: The state of the art. Adv. Colloid Interface Sci. 2014;205:187–
206. doi: 10.1016/j.cis.2013.11.018. [PubMed] [CrossRef] [Google Scholar]

15. Mujoriya R.Z., Dhamande K., Bodla R.B. Niosomal drug delivery system—A review. Int. J.
App. Pharm. 2011;3:7–10. [Google Scholar]

16. Ezzat H.M., Elnaggar Y.S.R., Abdallah O.Y. Improved oral bioavailability of the anticancer
drug catechin using chitosomes: Design, in-vitro appraisal and in-vivo studies. Int. J.
Pharm. 2019;565:488–498. doi: 10.1016/j.ijpharm.2019.05.034. [PubMed]
[CrossRef] [Google Scholar]

17. Manca M.L., Manconi M., Valenti D., Lai F., Loy G., Matricardi P., Fadda A.M. Liposomes
coated with chitosan–xanthan gum (chitosomes) as potential carriers for pulmonary delivery
of rifampicin. J. Pharm. Sci. 2012;101:566–575. doi: 10.1002/jps.22775. [PubMed]
[CrossRef] [Google Scholar]

18. Mertins O., Dimova R. Binding of Chitosan to Phospholipid Vesicles Studied with
Isothermal Titration Calorimetry. Langmuir. 2011;27:5506–5515.
doi: 10.1021/la200553t. [PubMed] [CrossRef] [Google Scholar]

19. Jain S., Jain V., Mahajan S.C. Lipid Based Vesicular Drug Delivery Systems. Adv.
Pharm. 2014;2014:1–12. doi: 10.1155/2014/574673. [CrossRef] [Google Scholar]

20. Mann J.F., Scales H.E., Shakir E., Alexander J., Carter K.C., Mullen A.B., Ferro V.A. Oral
delivery of tetanus toxoid using vesicles containing bile salts (bilosomes) induces significant
systemic and mucosal immunity. Methods. 2006;38:90–95.
doi: 10.1016/j.ymeth.2005.11.002. [PubMed] [CrossRef] [Google Scholar]

21. Yang L., Tucker I.G., Østergaard J. Effects of bile salts on propranolol distribution into
liposomes studied by capillary electrophoresis. J. Pharm. Biomed. Anal. 2011;56:553–559.
doi: 10.1016/j.jpba.2011.06.020. [PubMed] [CrossRef] [Google Scholar]

22. Rai S., Pandey V., Rai G. Transfersomes as versatile and flexible nano-vesicular carriers in
skin cancer therapy: The state of the art. Nano Rev. Exp. 2017;8:1325708.
doi: 10.1080/20022727.2017.1325708. [PMC free article] [PubMed] [CrossRef] [Google
Scholar]

23. Cevc G., Schatzlein A.G., Richardsen H. Ultradeformable lipid vesicles can penetrate the
skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM
experiments and direct size measurements. Biochim. et Biophys. Acta (BBA)-
Biomembr. 2002;1564:21–30. doi: 10.1016/S0005-2736(02)00401-7. [PubMed]
[CrossRef] [Google Scholar]
24. Cevc G. Lipid vesicles and other colloids as drug carriers on the skin. Adv. Drug Deliv.
Rev. 2004;56:675–711. doi: 10.1016/j.addr.2003.10.028. [PubMed] [CrossRef] [Google
Scholar]

25. Rother M., Lavins B.J., Kneer W., Lehnhardt K., Seidel E.J., Mazgareanu S. Efficacy and safety
of epicutaneous ketoprofen in Transfersome (IDEA-033) versus oral celecoxib and placebo in
osteoarthritis of the knee: Multicentre randomised controlled trial. Ann. Rheum.
Dis. 2007;66:1178–1183. doi: 10.1136/ard.2006.065128. [PMC free article] [PubMed]
[CrossRef] [Google Scholar]

26. Kumar A., Pathak K., Bali V. Ultra-adaptable nanovesicular systems: A carrier for systemic
delivery of therapeutic agents. Drug Discov. Today. 2012;17:1233–1241.
doi: 10.1016/j.drudis.2012.06.013. [PubMed] [CrossRef] [Google Scholar]

27. Fesq H., Lehmann J., Kontny A., Erdmann I., Theiling K., Rother M., Ring J., Cevc G., Abeck D.
Improved risk-benefit ratio for topical triamcinolone acetonide in Transfersome in
comparison with equipotent cream and ointment: A randomized controlled trial. Brit. J.
Dermatol. 2003;149:611–619. doi: 10.1046/j.1365-2133.2003.05475.x. [PubMed]
[CrossRef] [Google Scholar]

28. Cevc G. Transfersomes: Innovative transdermal drug carriers. In: Rathbone M.J., Hadgraft
J., Roberts M.S., editors. Modified-Release Drug Delivery Technology. 1st ed. Volume 126.
Marcel Dekker, Inc.; New York, NY, USA: 2002. pp. 533–546. [Google Scholar]

29. Touitou E. Composition of Applying Active Substance to or Through the Skin.

5540934A. U.S. Patent. 1996 Jul 30;

30. Touitou E., Dayan N., Bergelson L., Godin B., Eliaz M. Ethosomes novel vesicular carriers
for enhanced delivery: Characterization and skin penetration properties. J. Control.
Release. 2000;65:403–418. doi: 10.1016/S0168-3659(99)00222-9. [PubMed]
[CrossRef] [Google Scholar]

31. Shah S.M., Ashtikar M., Jain A.S., Makhija D.T., Nikam Y., Gude R.P., Steiniger F., Jagtap A.A.,
Nagarsenker M.S., Fahr A. LeciPlex, invasomes, and liposomes: A skin penetration study. Int. J.
Pharm. 2015;490:391–403. doi: 10.1016/j.ijpharm.2015.05.042. [PubMed]
[CrossRef] [Google Scholar]

32. Qadri G.R., Ahad A., Aqil M., Imam S.S., Ali A. Invasomes of isradipine for enhanced
transdermal delivery against hypertension: Formulation, characterization, and in vivo
pharmacodynamic study. Artif. Cells Nanomed. Biotechnol. 2016;45:139–145.
doi: 10.3109/21691401.2016.1138486. [PubMed] [CrossRef] [Google Scholar]

33. Walve J.R., Bakliwal S.R., Rane B.R., Pawar S.P. Transfersomes: A surrogated carrier for
transdermal drug delivery system. Int. J. Appl. Biol. Pharm. Technol. 2011;2:204–213. [Google
Scholar]

34. Sivannarayana P., Rani A.P., Saikishore V., VenuBabu C., SriRekha V. Transfersomes: Ultra
deformable vesicular carrier systems in transdermal drug delivery system. Res. J. Pharm. Dos.
Forms Technol. 2012;4:243–255. [Google Scholar]
35. Sachan R., Parashar T., Soniya S.V., Singh G., Tyagi S., Patel C., Gupta A. Drug carrier
transfersomes: A novel tool for transdermal drug delivery system. Int. J. Res. Dev. Pharm. Life
Sci. 2013;2:309–316. [Google Scholar]

36. Li J., Wang X., Zhang T., Wang C., Huang Z., Luo X., Deng Y. A review on phospholipids and
their main applications in drug delivery systems. Asian J. Pharm. Sci. 2015;10:81–98.
doi: 10.1016/j.ajps.2014.09.004. [CrossRef] [Google Scholar]

37. Bhasin B., Londhe V.Y. An overview of transfersomal drug delivery. Int. J. Pharm. Sci.
Res. 2018;9:2175–2184. doi: 10.13040/IJPSR.0975-8232.9(6).2175-84. [CrossRef] [Google
Scholar]

38. Lei W., Yu C., Lin H., Zhou X. Development of tacrolimus-loaded transfersomes for deeper
skin penetration enhancement and therapeutic effect improvement in vivo. Asian J. Pharm.
Sci. 2013;8:336–345. doi: 10.1016/j.ajps.2013.09.005. [CrossRef] [Google Scholar]

39. Pandey A. Role of surfactants as penetration enhancer in transdermal drug delivery

system. J. Mol. Pharm. Org. Process. Res. 2014;2:1–10. doi: 10.4172/2329-
9053.1000113. [CrossRef] [Google Scholar]

40. Duangjit S., Opanasopit P., Rojanarata T., Ngawhirunpat T. Characterization and in vitro
skin permeation of meloxicam-loaded Liposomes versus Transfersomes. J. Drug
Deliv. 2010;2011:1–9. doi: 10.1155/2011/418316. [PMC free article] [PubMed]
[CrossRef] [Google Scholar]

41. Aggarwal N., Goindi S. Preparation and evaluation of antifungal efficacy of griseofulvin
loaded deformable membrane vesicles in optimized guinea pig model of Microsporum canis
—Dermatophytosis. Int. J. Pharm. 2012;437:277–287.
doi: 10.1016/j.ijpharm.2012.08.015. [PubMed] [CrossRef] [Google Scholar]

42. Chen J., Lu W.-L., Gu W., Lu S.-S., Chen Z.-P., Cai B.-C. Skin permeation behavior of elastic
liposomes: Role of formulation ingredients. Expert Opin. Drug Deliv. 2013;10:845–856.
doi: 10.1517/17425247.2013.779252. [PubMed] [CrossRef] [Google Scholar]

43. Duangjit S., Opanasopit P., Rojanarata T., Ngawhirunpat T. Effect of edge activator on
characteristic and in vitro skin permeation of meloxicam loaded in elastic liposomes. Adv.
Mater. Res. 2011;194:537–540. doi: 10.4028/www.scientific.net/AMR.194-
196.537. [CrossRef] [Google Scholar]

44. Jacob L., Anoop K.R. A review on surfactants as edge activators in ultradeformable vesicles
for enhanced skin delivery. Int. J. Pharma Bio Sci. 2013;4:337–344. [Google Scholar]

45. Kim B., Cho H.-E., Moon S.H., Ahn H.-J., Bae S., Cho H.-D., An S. Transdermal delivery
systems in cosmetics. Biomed. Dermatol. 2020;4:1–12. doi: 10.1186/s41702-019-0053-
z. [CrossRef] [Google Scholar]

46. Kumar G.P., Rajeshwarrao P. Nonionic surfactant vesicular systems for effective drug
delivery—An overview. Acta Pharm. Sin. B. 2011;1:208–219.
doi: 10.1016/j.apsb.2011.09.002. [CrossRef] [Google Scholar]