A

PROJECT REPORT
ON

“TRANSFEROSOMES: A NEXT GENERATION

CARRIER FOR TRANSDERMALDRUG DELIVERY”

Subject –Practice School Paper Code –BP706PS

Submitted By:
Aman Kumar Yadav
Roll No.: 1220781015
B. Pharm. 7th Semester

Under supervision of: Submitted to:

Mr. Shivam Verma Prof.(Dr.) Rajiv Gupta
Assistant Professor Principal & Dean
School of Pharmacy School Of Pharmacy
BBD University BBD University

School Of Pharmacy

Babu Banarasi Das University, Lucknow

Academic session: 2025-26

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 CERTIFICATE

This certifies that Aman Kumar Yadav has successfully completed the project work
entitled “Transferosomes: A Next Generation Carrier For Transdermal Drug Delivery”
carried out at School Of Pharmacy, Babu Banarsi Das University, Lucknow under the
supervision of Mr. Shivam Verma Sir (Assistant Professor) during the academic session
2025-2026.

This project work has been carried out in partial fulfillment of the requirements for the award
of the degree of Bachelor Of Pharmacy, School Of Pharmacy, BBDU.

We hereby acknowledge the sincere effort and dedication put forth by the student in
completion of the project.

Date: Name & Designation of Supervisor:

Mr. Shivam Verma

Assistant Professor

School of Pharmacy(BBDU)

Place: Lucknow Signature:

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 ACKNOWLEDGEMENT

I would like to express my sincere gratitude to Mr. Shivam Verma Sir (Assistant Professor)
School of Pharmacy (BBDU), my project guide, for his invaluable guidance, encouragement,
and constant support throughout the course of this project. His insightful suggestions and
constructive feedback have been instrumental in the successful completion of this work.

I am also deeply thankful to Prof. (Dr.) Rajiv Gupta, Principal & Dean, of
School Of Pharmacy, BBDU, for providing me with the necessary resources and facilities to
carry out this project.

I extend my heartfelt thanks to all the faculty members and staff of the School Of Pharamcy,
Babu Banarasi Das University, Lucknow, for their cooperation and support.

I am especially grateful to my peers and friends for their motivation and helpful discussions,
which made this journey smoother and more enriching.

Finally, I owe my deepest gratitude to my family for their unwavering encouragement,

patience, and moral support throughout the course of this project.

Date: Name : Aman Kumar Yadav

Place: Lucknow Signature:

DECLARATION

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 I hereby declare that the project work entitled “Transferosomes: A Next Generation
Carrier For Transdermal Drug Delivery” submitted by me to School Of Pharamcy, Babu
Banarasi Das University, Lucknow, in partial fulfillment of the requirements for the award
of the degree of Bachelor of Pharmacy, is a record of my original work carried out under the
guidance of Mr. Shivam Verma Sir (Assistant Professor) School of Pharmacy (BBDU).

I further declare that this project work has not been submitted previously, in part or full, for
the award of any degree, diploma, or fellowship, and that the work is entirely my own except
where due reference has been made.

Date: Name: Aman Kumar Yadav

Place: Lucknow Signature:

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 Abstract

Transferosomes is a carrying body for targeted transdermal drug delivery system. This are
special types of liposomes, consisting of phosphatidylcholine and an edge activator. This
system also takes advantage of phospholipids vesicles as transdermal drug carrier. It
penetrate the stratum corneum by either intracellular route or the transcellular route by the
generation of “osmotic gradient”.

Advantages of Transferosomes are wide range of solubilities, better penetration,

biocompatible and biodegradable etc. Advantages of Transferosomes are oxidative
degradation, expensive, etc. The transfersomes were formulated by the conventional rotary
evaporation sonication method. It contains phospholipids, surfactant and the drug were
formulated. Evaluation parameters of transferosome are as Vesicle size distribution and zeta
potential, Vesicle morphology, No. of vesicles per cubic mm, Entrapment efficiency, Drug
content, Turbidity measurement, Degree of deformability or permeability measurement,
Penetration ability, Occlusion effect, Surface charge and charge density, In-vitro drug release,
in-vitro Skin permeation Studies, Physical stability.

Transferosomes can be applied in controlled release, transportation of large molecules

weight compounds, target delivery to peripheral subcutaneous tissues, transdermal
immunization etc.

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 INDEX

[Link]. Description [Link].

1. Introduction 7-10

2. Advantages of Transferosomes 10-11

3. Disadvantages of Transferosomes 11

4. Mechanism of Transport 12-13

5. Material for Transferosomes 14-15

6. Method of preparation 15-16

7. Stability of transferosomes 16

8. Characterizations 16-17

9. Evaluations of Transferosomes 17-20

10. Safety considerations 20

11. Applications of Transferosomes 20-22

12. Transferosomes for transcutaneous vaccination 22-23

13. Marketed transferosomes and those in clinical trials 23-24

14. Transferosomes for phytochemical agents 24-25

15. Integration transferosomes with artificial intelligence 26

16. Future perspectives 27

17. Conclusion 27

18. References 28-33

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 1. Introduction
Transfersome is a trademark registered by the German company IDEA AG, which refers to
its proprietary drug delivery technology.

The name means “carrying body” and is derived from the Latin word 'transferre', meaning 'to
carry across' and the Greek word 'soma', meaning 'a body'.(1,2)

A transfersome carrier is an artificial vesicle designed to exhibit the characteristics of a cell

vesicle or a cell engaged in exocytosis, and thus suitable for controlled and potentially,
targeted drug delivery. Transfersomes are complex vesicles that have extremely flexible &
self-regulating membranes, which make the vesicle very deformable. Transfersome vesicle
can cross microporous barriers efficiently, even if the porous are much smaller than the
vesicles size.(1,2,3)

Figure.1

Drug Delivery via the route is an interesting option in this respect because transdermal route
is convenient and safe. They offers several advantages over conventional drug delivery
system like avoidance of first pass metabolism, predictable and extended duration of action,
minimizing undesirable side effects, utility of short half-life drugs, improving physiological
and pharmacological response, avoiding the fluctuation in drug levels, inter-and intra-patient
variations and most importantly, it provides patients convenience.

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Despite major research and development efforts in transdermal systems and the advantages
of these routes, low stratum corneum permeability limits the usefulness of topical drug
delivery.

To overcome this, various methods have been assessed to increase stratum corneum
permeability.

To date many physical and chemical approaches have been applied to increase the efficacy
of the material to transfer across the intact skin, by use of the penetration enhancers,
iontophoresis, iontophoresis and the use of colloidal carriers such as lipid vesicles (liposome
and proliposomes) and non-ionic surfactant vesicles (noisome and proniosomes).

Vesicular system used in transdermal drug delivery such as liposomes, niosomes, or

microemulsions usually remains confined to the skin surface and therefore do not transport
drugs efficiently through the skin.

By using the concept of rational membrane design a special type of composite bodies, so-
called transfersomes have been developed, which overcome the filtration problem and
penetrate the skin barrier along the transcutaneous gradient.

Transfersomes is recent novel drug delivery system and are special types of liposome,
consisting of phosphatidylcholine and an edge activator. This system also takes advantage of
phospholipids vesicles as transdermal drug carrier. They are self-optimized aggregates, with
the ultra flexible membrane, which deliver the drug reproducibly either into or through the
skin. The system delivers the drug with high efficiency depending on the choice of
administration or application. This system has several order magnitude of elasticity and
flexibility over liposomal drug delivery which makes it favorable for efficient skin
penetration and hence for the novel drug delivery system. They overcome the skin
penetration difficulty by squeezing themselves along the intracellular sealing lipid of the
stratum corneum. (1,2,3,4)

With the application of mechanical stress, they can enter through stratum corneum in self
adapting manner because of their high vesicle deformability. Flexibility or elasticity of
transfersomes membrane is achieved by mixing suitable surface-active components (edge
activator) in the proper ratios. The resulting flexibility of transfersomes membrane minimizes
the risk of complete vesicle rupture in the skin and allows them to follow the natural water
gradient across the epidermis, when applied under nonocclusive condition.

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They can penetrate the intact stratum corneum spontaneously by either intracellular lipid or
transcellular route. The high and self-optimizing deformability of typical composite bodies
membrane, which are adaptable to ambient stress allow the ultra deformable transfersomes to
change its membrane composition locally and reversibly, when it is pressed against or
attracted into narrow pore. When applied on the skin, the carrier searches and exploits
hydrophilic pathways or pores between the cells, where it opens wide enough to permit the
entire vesicle to pass through stratum corneum along with drug molecule, deforming itself
extremely to accomplish this without losing its vesicular integrity. This enables them to cross
various transport barriers efficiently.(2,4)

Transfersomes are ultra deformable, self optimized aggregates for transdermal application
containing a mixture of lipids and biocompatible membrane softeners. Though basic
organization is broadly similar to a liposome, the transfersome differs by its softer, more
deformable and better adjustable artificial membrane they posses.

Transfersome penetrate the stratum corneum by either intracellular route or the transcellular
route by the generation of “osmotic gradient” due to evaporation of water. Thus a
transferosome vesicle, when applied on an open biological surface, such as non occluded
skin, tends to penetrate its barrier and migrate into the water-rich deeper strata to secure
adequate hydration. As the vesicles are elastic, they can squeeze through the pores in stratum
corneum (though these pores are less than one-tenth of the diameter of vesicles).

Transfersome vesicles can transport molecules that are too big to diffuse through skin Eg:
systemic delivery of therapeutically meaningful amounts of macromolecules, such as insulin
or interferon. Other applications include the transport of small molecule drugs which have
certain physicochemical properties which would otherwise prevent them from diffusing
across the barrier.

Now a day, transfersome can be used to target peripheral subcutaneous tissue. The Non-
steroidal anti-inflammatory drug (NSAID) ketoprofen in a transfersome formulation in the
trade mark Diractin gained marketing approval by the Swiss regulatory agency (Swiss Medic)
in 2007.

Topical immunization using cationic transfersomes based DNA vaccine offers all the
advantages of DNA vaccines and in addition overcome the disadvantages of classical
invasive methods of vaccination. Liposomal as well as niosomal systems, are not suitable for

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transdermal delivery, because of their poor skin permeability, breaking of vesicles, leakage of
drug, aggregation and fusion of vesicles.

To overcome these problems, a new type of carrier system called "Transfersome", has
recently been introduced, which is capable of transdermal delivery of low as well as high
molecular weight drugs. Transferosomes are specially optimized, ultra deformable (ultra
flexible) lipid supra molecular aggregates, which are able to penetrate the mammalian skin
intact.

The name means “carrying body”, and is derived from the Latin word 'transferre', meaning
“to carry across” and the Greek word “soma”, for a “body”. A transferosome carrier is an
artificial vesicle designed to be like a cell vesicle or a cell engaged in exocytosis, and thus
suitable for controlled and potentially targeted, drug delivery.(1,2,3,4)

2. Advantages of Transferosomes

 Transfersomes possess an infrastructure consisting of hydrophobic and hydrophilic

moieties together and as a result can accommodate drug molecules with wide range of
solubilities. They can deform and pass through narrow constriction (from 5 to 10
times less than their own diameter) without significant loss.(5)
 High deformability of this system gives better penetration of intact vesicles. They can
act as a carrier for low as well as high molecular weight drugs e.g. analgesic,
anaesthetic, corticosteroids, sex hormone, anticancer, insulin and albumin. (6)
 They are biocompatible and biodegradable as they are made from natural
phospholipids similar to liposomes.
 They have high entrapment efficiency, in case of lipophilic drug near to 90%.
 They protect the encapsulated drug from metabolic degradation example: protein and
peptides.
 They act as depot, releasing their contents slowly and gradually & can be used for
both systemic as well as topical delivery of drug. They are easy to scale up, as
procedure is simple and avoid unnecessary use or pharmaceutically unacceptable
additives.
 At first glance, transfersomes appear to be remotely related to lipid bilayered vesicle,
liposomes. However in functional terms, transfersomes differ vastly from commonly
used liposomes in that they are much more flexible and adaptable.

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  The extremely high flexibility of their membrane permits transfersomes to squeeze
themselves even through pores much smaller than their own diameter.
 Transfersomes possess an infrastructure consisting of hydrophobic and hydrophilic
moieties together and as a result, can accommodate drug molecules with wide range
of solubility.
 Transfersomes can deform and pass through narrow constriction (from 5 to 10 times
less than their own diameter) without measurable loss. This high deformability gives
better penetration of intact vesicles through tight junctions.
 They can act as a carrier for low as well as high molecular weight drugs e.g.
analgesic, anesthetic, corticosteroids, sex hormone, anticancer, insulin, gap junction
protein and albumin. They are biocompatible and biodegradable as they are made
from natural phospholipids similar to liposomes. They have high entrapment
efficiency, in case of lipophilic drug close to 90%.
 They protect the encapsulated drug from metabolic degradation. They act as depot,
releasing their contents slowly and gradually. They can be used for both systemic as
well as topical delivery of drug. Easy to scale up, as procedure is simple, do not
involve lengthy procedure and unnecessary use or pharmaceutically unacceptable
additives.(5,6)

3. DISADVANTAGES

 Transfersomes are chemically unstable because of their predisposition to oxidative

degradation. (6)
 Purity of natural phospholipids is another criteria militating against adoption of
transfersomes as drug delivery vehicles.
 Transfersomes formulations are expensive.

4. MECHANISM OF TRANSPORT

Transferosomes overcome the skin penetration difficulty by squeezing themselves along the
intracellular sealing lipids of stratum corneum. At present, the mechanism of enhancing the
delivery of active substances in and across the skin is not very well known.

11
Two mechanisms of action have been proposed:

1. Transferosomes act as drug vectors, remaining intact after entering the skin.
(8,10)

Figure :02 Intercellular transport of transferosomes through SC into the dermis

2. Transferosomes act as penetration enhancers, disrupting the highly organized

intercellular lipids from stratum corneum and therefore facilitating the drug
molecules penetration in and across the stratum corneum.(9)

The transfersomes vesicles usage in drug delivery consequently relies on the carrier‟s ability
to widen and overcome the hydrophilic pores in the skin. Intracellular drug transportation
may involve diffusion of vesicle lipid bilayer with the cell membrane like normal
endocytosis. The mechanism is thus comlplex and involves advanced principles of mechanics
combined with material transport and hydration/osmotic force.

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 Figure: 03 Dermal kinetics of transferosomes

A schematic overview of different stages of the transferosomes when applied to the skin. On
the surface of the skin, the transferosomes mainly take part in three interactions:

 Transferosome-transferosome interaction,
 Transferosome-drug interaction, and
 Transferosome-skin interaction.

The transferosome-skin interaction results in the diffusion of transferosome through the SC

using intercellular route. The diffusion of the transferosomes is driven by the water gradient
in the skin layers (hydrotaxis). When transferosome reaches the viable epidermis, the drug is
released out of the transferosome into the aqueous environment. The released drug is then
taken up into the systemic circulation by the capillary bed in the dermis.(8,9,10)

Possible pathways for a penetrant to cross the skin barrier:

(1) Across the intact horny layer,

(2) Through the hair follicles with the associated sebaceaous glands, or

(3) Via the sweat glands(7,8,9,10)

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5. MATERIAL FOR TRANSFEROSOMES

Transferosomes is a self adaptable and optimized mixed lipid aggregate and composed of
phospholipids like phosphatidyl choline which self assembles into lipid bilayer in aqueous
environment and closes to forma vesicle. A bilayer softening component (such as a
biocompatible surfactant or an amphiphile drug) is added to increase lipid bi layer flexibility
and permeability. This second component is called as edge activator. (11,12)

An edge activator consists usually of single chain surfactant that causes destabilization of the
lipid bilayer thereby increasing its fluidity and elasticity. The newer elastic vesicles were
introduced by Van den berg in 1998, consisting of non ionic surfactant as the edge activator
30. Flexibility of tranferosomes membrane can be altered by mixing suitable surface active
agents in the proper ratios. The resulting, flexibility and permeability optimized,
transferosome vesicle can therefore adapt its shape to surrounding stress easily and rapidly,
by adjusting local concentration of each bi layer component to the local stress experienced by
the bi layer. This flexibility also minimizes the risk of complete vesicle rupture in the skin
and allows transferosomes to follow the natural water gradient across the epidermis, when
applied under non occlusive condition. Vesicles composed of phospholipids as the main
ingredient( soyaphosphatidylcholine,egg phosphatidylcholine, dipalmitylphosphatidylcholine,
etc), 10-25% surfactant for providing flexibility (ethanol, methanol) and hydrating medium
consisting of saline phosphate buffer (pH 6.5-7). Dye like Rhodamine 123, Nile red for
Confocal Scanning Laser Microscopy.(12,14)

Materials commonly used for the preparation of transfersomes are summarized in table
below:(11,12,13,14)

Ingredient Examples Functions

Soya Phosphatidylcholine
Phospholipid Egg Phosphatidylcholine Vesicle forming Componet
Disteryl
Phosphatidylcholine
Sodium Cholate
Surfactant Sodium deoxy Cholate For Providing Flexibility
Tween 80
Span 80
Ethanol
Alcohol Methanol As a Solvent
Rhodamine-123

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 Rhodamine-DHPE For Confocal ScaningLaseer
Dye Flurescein-DHPE Microscopy (CSLM) Study
Nil red
6 Corboxy fluorescence
Saline phosphate buffer
Buffering Agent (PH 6.5)
7% v/v ethanol As a hydrating medium
Tris buffer ( PH 6.5)

6. METHOD OF PREPARATION

A. Thin film hydration technique is employed for the preparation of transfersomes

which comprised of three steps:

1. A thin film is prepared from the mixture of vesicles forming ingredients that is
phospholipids and surfactant by dissolving in volatile organic solvent (chloroform methanol).
Organic solvent is then evaporated above the lipid transition temperature (room temp. for
pure PC vesicles, or 50°C for dipalmitoyl phosphatidyl choline) using rotary evaporator.
Final traces of solvent were removed under vacuum for overnight.

2. A prepared thin film is hydrated with buffer (pH 6.5) by rotation at 60 rpm for 1 hr at the
corresponding temperature. The resulting vesicles were swollen for 2 hr at room temperature.

3. To prepare small vesicles, resulting vesicles were sonicated at room temperature or 50°C
for 30 min. using a bath sonicator or probe sonicated at 4°C for 30 min. The sonicated
vesicles were homogenized by manual extrusion 10 times through a sandwich of 200 and 100
nm polycarbonate membranes. (11,14)

B. Modified hand shaking, lipid film hydration technique is also founded for the
preparation of transfersomes which comprised following steps

1. Drug, lecithin (PC) and edge activator were dissolved in ethanol: chloroform (1:1)
mixture. Organic solvent was removed by evaporation while hand shaking above lipid
transition temperature (43°C). A thin lipid film was formed inside the flask wall with
rotation. The thin film was kept overnight for complete evaporation of solvent.

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2. The film was then hydrated with phosphate buffer (pH 7.4) with gentle shaking for 15
minute at corresponding temperature. The transfersome suspension further hydrated up to 1
hour at 2-8°C. (11,12,13,14)

7. STABILITY OF TRANSFEROSOMES

Transferosomes are chemically unstable because of their predisposition to oxidative

degradation. Purity of natural phospholipids is another criterion militating against adoption of
transferosomes as drug delivery vehicles. Transferosome formulations are expensive.

8. CHARACTERIZATION

• Visualization of transferosomes can be performed using transmission electron microscopy

(TEM) and scanning electron microscopy (SEM).(19)

• Particle size and size distribution can be determined by dynamic light scattering (DLS) and
photon correlation spectroscopy (PCS).(19,20)

• The drug entrapment efficiency by transferosomes can be measured by the

ultracentrifugation technique.

• Vesicle stability can be determined by assessing the size and structure of the vesicles over
time, and drug content can be quantified by high performance liquid chromatography (HPLC)
or Spectrophotometric methods.

• In vitro drug release can be measured using a diffusion cell or a dialysis method .(21,22)

• Propensity of penetration[23]

• The magnitude of the transport driving force, of course, also plays an important role:

Flow = Area × (Barrier) Permeability × (Trans-barrier) force.

Therefore, the chemically driven lipid flow across the skin always decreases dramatically
when lipid solution is replaced by some amount of lipids in a suspension.

Encapsulation efficiency is expressed as the percent of drug trapped:(21)

% Entrapment = Total drug - Diffused drug × 100

Total drug

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 Parameter Method
Vesicle shape morphology Transmission electron microscopy
Entrapment efficiency Mini column centrifugation method
Vesicle size and size distribution Dynamic light scattering method
Skin permeation potential Confocal laser scanning microscopy
Phospholipid surfactant interaction Fluorescence microscopy
Degree of deformability Transmission electron microscopy
Surface charge and charge density Thin layer chromatography
Methods for the characterization of transferosomes:

In vitro drug release study Differential scanning calorimeter

Effect on the skin structure Zeta meter, Nephelometer
Stability study Side by side diffusion cell with artificial or
biological membrane,dialysis bag diffusion,
Histological studies, Transmission electron
microscope, Dynamic light scattering method

9. EVALUATION OF TRANSFEROSOMES

1. Vesicle size distribution and zeta potential

Vesicle size, size distribution and zeta potential were determined by Dynamic Light
Scattering system by Malvern Zeta sizer.(17)
2. Vesicle morphology: World Journal of Pharmacy and Pharmaceutical Sciences
Vesicle diameter can be determined using photon correlation spectroscopy or dynamic
light scattering (DLS) method. Samples were prepared in distilled water, filtered
through a 0.2 mm membrane filter and diluted with filtered saline and then size
measurement done by using photon correlation spectroscopy or dynamic light
scattering (DLS) measurements. Transfersomes vesicles can be visualized by TEM,
phase contrast microscopy, etc. The stability of vesicle can be determined by
assessing the size and structure of vesicles over time. Mean size is measured by DLS
and structural changes are observed by TEM.(16)

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3. No. of vesicles per cubic mm
This is an important parameter for optimizing the composition and other process
variables. Non sonicated Transfersome formulations are diluted five times with 0.9%
sodium chloride solution. Haemocytometer and optical microscope can then be used
for further study. The Transfersomes in 80 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.
4. Entrapment efficiency
The entrapment efficiency is expressed as the percentage entrapment of the drug
added. Entrapment efficiency was determined by first separation of the un-entrapped
drug by use of mini-column centrifugation method. After centrifugation, the vesicles
were disrupted using 0.1% Triton X-100 or 50% n-propanol]. The entrapment
efficiency is expressed as: Entrapment efficiency = (Amount entrapped / Total
amount added) ×100
5. Drug content
The drug content can be determined using one of the instrumental analytical methods
such as modified high performance liquid chromatography method (HPLC) method
using a UV detector, column oven, auto sample, pump and computerized analysis
program depending upon the analytical method of the pharmacopoeial drug.
6. Turbidity measurement
Turbidity of drug in aqueous solution can be measured using nephelometer.
7. Degree of deformability or permeability measurement
In the case of transfersomes, the permeability study is one of the important and unique
parameter for characterization. The deformability study is done against the pure water
as standard. Transfersomes preparation is passed through a large number of pores of
known size (through a sandwich of different micro porous filters, with pore diameter
between 50 nm and 400 nm, depending on the starting transfersomes suspension).
Particle size and size distributions are noted after each pass by dynamic light
scattering (DLS) measurements.
8. Penetration ability
Penetration ability of Transfersomes can be evaluated using fluorescence microscopy.
9. Occlusion effect

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 Occlusion of skin is considered to be helpful for permeation of drug in case of
traditional topical preparations. But the same proves to be detrimental for elastic
vesicles. Hydrotaxis (movement in the direction) of water is the major driving force
for permeation of vesicles through the skin, from its relatively dry surface to water
rich deeper regions. Occlusion affects hydration forces as it prevents evaporation of
water from skin.
10. Surface charge and charge density
Surface charge and charge density of Transfersomes can be determined using zeta
sizer.
11. In-vitro drug release
In vitro drug release study is performed for determining the permeation rate. Time
needed to attain steady state permeation and the permeation flux at steady state and
the information from in vitro studies are used to optimize the formulation before more
expensive in vivo studies are performed. For determining drug release, transfersomes
suspension is incubated at 320C and samples are taken at different times and the free
drug is separated by mini column centrifugation. The amount of drug released is then
calculated indirectly from the amount of drug entrapped at zero times as the initial
amount (100% entrapped and 0% released).
12. In-vitro Skin permeation Studies
Modified Franz diffusion cell with a receiver compartment volume of 50ml and
effective diffusion area of 2.50 cm2 was used for this study. In vitro drug study was
performed by using goat skin in phosphate buffer solution (pH 7.4). Fresh Abdominal
skin of goat were collected from slaughterhouse and used in the permeation
experiments. Abdominal skin hairs were removed and the skin was hydrated in
normal saline solution. The adipose tissue layer of the skin was removed by rubbing
with a cotton swab. Skin was kept in isopropyl alcohol solution and stored at 0-40˚C.

To perform skin permeation study, treated skin was mounted horizontally on the receptor
compartment with the stratum corneum side facing upwards towards the donor
compartment of Franz diffusion cell. The effective permeation area of donor compartment
exposed to receptor compartment was 2.50cm2 and capacity of receptor compartment was
50ml. The receptor compartment was filled with 50ml of phosphate buffer (pH 7.4) saline
maintained at 37 ± 0.5˚C and stirred by a magnetic bar at 100RPM. Formulation

19
 (equivalent to 10mg drug) was placed on the skin and the top of the diffusion cell was
covered. (16,17)

13. Physical stability

The initial percentage of the drug entrapped in the formulation was determined and
were stored in sealed glass ampoules. The ampoules were placed at 4 ± 20C months.
Samples from each ampoule were analyzed after 30 days to determine drug leakage.
Percent drug lose was calculated by keeping the initial entrapment of drug as 100%.

10. SAFETY CONSIDERATIONS

Phospholipid suspensions comprising liposomes have been reported to be harmless

and non-irritating to the skin after repeated epicutaneous administration; they may
even have additional advantageous cosmetic effect.
The main component of transferosomes is typically soya phosphatidylcholine of
greater than 95% purity, which is generally regarded as safe because it has been
already in use as an emulsifier in micro emulsion for the parenteral nutrition and is
also used in injectable drug information.
In light of these data, one can expect the transferosome product to be very safe from
the carrier point of view.[22]

11. APPLICATION OF TRANSFEROSOMES

1. Transfersomes have the potential for the controlled release of the administered
drug and increasing the stability of labile drugs due to the incorporation of
phospholipids(18)
2. Large molecules weight compounds can be easily transported across the skin
with the help of transfersomes. For example, insulin, interferon like leukocytic
derived interferon (INF) can be delivered through mammalian skin. They have
been widely used as a carrier for the transport of other proteins and peptides. As
protein sand peptides are large biogenic molecules difficult to transport into the
body and degraded in the GI tract and transdermal suffers due to their large size.

20
3. since transfersomes obtain similar bioavailability to subcutaneous injection.
Human serum albumin was found to be effective in producing the immune
response when delivered by transdermal route encapsulated in Transfersomes.
4. Peripheral drug targeting: the ability of transfersomes to target peripheral
subcutaneous tissues is due to minimum carrier associated drug clearance through
blood vessels in the subcutaneous tissue.

5. Transdermal immunization: Transcutaneous hepatitis-B vaccines have given good

results. A 12 times higher AUC was obtained for zidovudine as compared to
normal control administration. Selectivity in deposition in RES (which is the
usual site for residence of HIV) was also increased.(18)

6. NSAIDS are associated with number of GI side effects. These can be overcome
by transdermal delivery using ultra deformable vesicles.

7. Transferosomes have been widely used as a carrier for the transport of proteins
and peptides. Proteins and peptide are large biogenic molecules which are very
difficult to transport into the body, when given orally they are completely
degraded in the GI tract. These are the reasons why these peptides and proteins
still have to be introduced into the body through injections. Various approaches
have been developed to improve these situations. The bioavaibility obtained from
transferosomes is somewhat similar to that resulting from subcutaneous injection
of the same protein suspension.(17)

8. The transferosomal preparations of this protein also induced strong immune

response after the repeated picutaneous application, for example the adjuvant
immunogenic bovine serum albumin in transferosomes, after several dermal
challenges is as active immunologically as is the corresponding injected proteo-
transferosomes preparations. (17)

9. Delivery of insulin by transferosomes is the successful means of non invasive

therapeutic use of such large molecular weight drugs on the skin. Insulin is
generally administered by subcutaneous route that is inconvenient. Encapsulation
of insulin into transferosomes (transfersulin) overcomes these entire problems.

21
 After transfersulin application on the intact skin, the first sign of system
ichypoglycemia are observed after 90 to 180 min, depending on the specific
carrier composition.

10. Transferosomes have also been used as a carrier for interferons, for example
INF-α is a naturally occurring protein having antiviral, anti proliferive and some
immunomodulatory effects. Transferosomes as drug delivery systems have the
potential for providing controlled release of the administered drug and increasing
the stability of labile drugs.

11. Another most important application of transferosomes is transdermal

immunization using transferosomes loaded with soluble protein like integral
membrane protein, human serum albumin and gap junction protein.

These approach offers at least two advantages, first they are applicable without injection and
second, they give rise to rather high titer and possibly, to relatively high IgA levels.
Transferosomes have also used for the delivery of corticosteroids. Transferosomes improves
the site specificity and overall drug safety of corticosteroid delivery into skin by optimizing
the epicutaneously administered drug dose.(17,18)

12. TRANSFEROSOMES FOR TRANSCUTANEOUS VACCINATION

Transferosomes can be used for targeted distribution to periphery submucosa, controlled drug
release, and transdermal vaccination. Transdermal vaccination is targeting distinctive but
dissimilar population of specialized antigen-presenting cells (APCs) present in the epidermis
and dermis (Figure 2). The innate “nonspecific” cutaneous immune system is of great
potential for transdermal vaccination, as optimum antigen presentation by professional APCs
is required to originate a strong adaptive immune response (24).

The deformable nature of transferosomes makes it a delivery candidate for transcutaneous

immunization (25). Their elasticity increases the skin permeability to antigens in the
existence of an osmotic gradient by squeezing across the intercellular regions of the integral
SC. compared to conventional liposomes, transferosome increases the potential for antigen
delivery to antigen-presenting cells (APCs) (26).

22
However, various studies have yielded conflicting results regarding its effectiveness in
improving skin permeability. For example, delivery of hepatitis B surface antigen (HBsAg)
plasmid DNA cationic complex by ultra-elastic cationic liposomes showed higher levels of
humoral and cellular immune responses in comparison to vaccination with regular liposomes
(27). Ding et al. reported transcutaneous immunization by edge-activated liposomes.

They did not improve immunogenicity but required the use of physical permeation enhancers
such as micro needles (28) successfully modified transferosomes loaded tetanus toxoid
antigen fabricated by Thin-Film Hydration with vesicles size of 180 nm (29). These
transferosomes were tested in vivo and complete immunization was achieved in mice with
tetanus toxoid. Chopra et al. analysis of cytokine distribution patterns in immunized mice
revealed of cutaneous immune response profile to transcutaneous antigen delivery via such
carriers.

Furthermore, their results confirm the efficacy of transferosomes as carrier to antigen

delivery and it can improve future noninvasive immunization procedures. It may result in the
development of vaccines which result in controlled release of cytokines, allowing
unprecedented studies of immune responses in intact organs (29).

[Link] TRANSFEROSOMESAND THOSE IN CLINICAL

TRIALS

Several formulations based on transferosomes are now being evaluated at various stages of
clinical studies (Table 1). A Phase III clinical trial concerning ketoprofen safety was
conducted. Here the drug was loaded into transferosomes and was found to be effective for
osteoarthritis treatment (32). The topical administration of insulinloaded transferosomes
(Transfersulin) for the treatment of diabetes is being examined. Transfersulin was
demonstrated in preclinical investigations to lower blood glucose levels in rabbits with
alloxan-induced diabetes (33). In 2007 Swiss Medic, successfully launched ketoprofen in a
transferosome formulation to the market for musculoskeletal pain. The trade name is
“Ketoprofen Transdermal,” manufactured by “IDEA AG” Pharmaceuticals Pvt. Ltd (34).

23
 Clinical Sample Drug
Study title trial size
phase
A double-blind, placebo-controlled evaluation of safety Phase II 360 ketoprofen
and efficacy of epicutaneously applied IDEA-033
(ketoprofen in transferosome) in comparison to oral
celecoxib for the treatment of pain associated with
osteoarthritis of the knee (30)
Multiple-dose, randomized, subject and observer blinded, Phase I 82 Ketoprofen
placebo-controlled, doubledummy study of epicutaneously
applied ketoprofen Transferosome® Gel with or without
combination with oral celecoxib for the treatment of
muscle pain induced by eccentric exercise (31)
Multicentre, randomized, double-blind, placebo-controlled Phase III 555 Ketoprofen
study of safety and efficacy of epicutaneously applied
Diractin® (Ketoprofen in Transferosome® Gel) for the
treatment of osteoarthritis of the knee (31)
Multicenter, randomized, double-blind, placebo- and Phase III 1399 Ketoprofen
active-controlled study of safety and efficacy of two
dosages of epicutaneously applied Diractin® (ketoprofen
in Transferosome® Gel) for the treatment of osteoarthritis
of the knee (32).
Table 1. Transferosomes based formulations in clinical trials

[Link] FOR PHYTOCHEMICAL AGENTS

Phytochemicals found in medicinal herbs are now frequently employed to treat various
ailments. However, their lack of selectivity, permeability and limited bioavailability may
impede their therapeutic utility (35). Transferosomes can aid in the overcoming the poor
bioavailability of such compounds. To date, several phytochemical agents have been
successfully encapsulated into transferosomes.

Abdallah et al., were able to design stable nano-transferosome for Silymarin gel for three
months to target hyperglycemia (35). In vivo studies revealed that the developed formulation
significantly reduced blood glucose level. Ex-vivo results showed a high transdermal flux
with the result of (92.41 μg/cm2 h). Abdallah et al. suggested that the improvement of skin
permeability might be due to the use of Span 80 a nonionic surfactant that improves

24
membrane fluidity (36). In addition to vesicle flexibility, it also facilitates drug diffusion and
interactions between surfactants and skin lipids.

Teucrium marum L. (Lamiaceae) is a phytochemical agent for skin lesion that has been
encapsulated into transferosomes by Firoznezhad et al., (37). With composition of soy
lecithin (S75, 180 mg/ mL), Teucrium marum extract (40 mg/mL) and Tween 80 (15
mg/mL). A 99 nm particle size was achieved with Polydispersity index (PDI) of 0.22. The
same composition was also used but with the addition of a combination of glycerol and water
(25:75) to attain a new form of transferosome now termed a “glycerosome.” The authors also
prepared “hyalurosomes” by dissolving sodium hyaluronate in water. These particles were
then adjusted by adding glycerol to the sodium hyaluronate aqueous solution at a ratio of
(25:75), producing “glycerohyalurosomes” a novel formulation system of Teucrium marum.
The achieved particle size was 111 and 104 nm, respectively. Based on the in vitro results
glycerohyalurosomes may be a promising vesicle as compared to the hyalurosome as delivery
system for Teucrium marum (37).

Honokiol is a natural chemical compound that demonstrates anticancer effects against

melanoma cells. Ezzeldean et al. effectively fabricated transferosomes loaded with honokiol
using a heating method with lecithin and the edge activator sodium deoxycholate (38). They
noted that the increase in the edge activator (sodium deoxycholate) produced reduced vesicle
elasticity due to the bulkiness of the steroid-like structure of the sodium deoxycholate and
less deformable vesicles (39).

Similarly, further phytochemical agents were developed in transferosome formulations such

as curcumin transferosomes in a gel formulation using Carbopol-934 as gelling agent. The
authors of this study were able to deliver curcumin into arthritic dermal tissue and were able
to treat complete Freud's adjuvant (CFA)-induced arthritis (40).

Furthermore, they were able to achieve a high entrapment efficiency of Curcuma comosa in
transferosomes containing phospholipids and Tween 80 at a ratio of 90% and 10%,
respectively. Other phytochemicals encapsulated in transferosomes include rose bengal (41),
Myrciaria jaboticaba (42), a natural chondroprotective agent Rhein (43), ratite oils (44),
resveratrol (45), mangiferin (46), sulforaphane (47), and sinomenine (48).

25
[Link] TRANSFEROSOMES WITH ARTIFICIAL
INTELLIGENCE

Defining the right composition ratio of the formulation may be time consuming but it is the
most crucial parameter to consider since it affects the physicochemical characteristics of the
transferosomes produced (50).

Integrating AI and machine learning into nanoscale drug evaluation and formulation is the
future and may expedite the highthroughput and rational design development of highly
efficient nanoformulated pharmaceuticals and smart materials with predefined functions (51).
AI algorithms only require a training dataset which can be created through automated
synthetic techniques or obtained from the literature. This can be achieved by a large scale of
datasets and no-large scale of datasets. A large-scale dataset allows the algorithm to build
accurate predictive models that then can be translated into an improved nanoformulation.
Artificial neural networks (ANN) can be used without a large set of data. Li et al. used ANN
poly lactic-co-glycolic acid (PLGA) nanoparticles (52 ). They reported that the use of ANN
decreased the prediction error from 28.0% to 2.93% for particle size and from 19.4% to
2.99% for drug release (52). The same approach can be followed to formulate transferosome
formulations.

Another group of authors used ANN with continuous genetic algorithms to study
formulation factors affecting particle size and entrapment efficiency of nanoparticles. They
were successfully able to identify the response with a minimum number of required runs to
formulate the optimum verapamilloaded nanoparticle (52).

More recently, Reker et al. utilized a random forest machine learning model to detect the
best pairing between drug and excipient. Out of 2.1 million pairs, 100 self-assembling drug
nanoparticles were obtained. Chemical substructures and physicochemical properties of both
drug and excipient besides showing the formation of micrometer-sized and polydisperse
structures in all cases were used to describe each drug-excipient pair. The model designed by
Reker et al. is a promising model to formulate nanocarriers with less time and higher loading
capacity (53). However, to advance the field further, pharmaceutical scientists and AI experts
have to work closely together to develop improved AI systems that produce accurate results
using automated robotic particle synthesis.

26
[Link] PERSPECTIVES

The high tolerability and efficiency of these vesicular systems open vast potential therapeutic
uses. These nanocarriers might offer advanced local and systemic new therapies with agents
that are unable to efficiently penetrate the stratum corneum via passive diffusion. The non
steroidal anti-inflammatory drug (NSAID), ketoprofen, in a transferosome formulation
gained marketing approval by the Swiss regulatory agency (SwissMedic). The product is
expected to be marketed under the trademark Diractin. Further therapeutic products based on
the transferosome technology, according to IDEA AG, are in clinical development .

[Link]

Transfersomes display deformable properties that improve the stability, penetration,

elasticity, and delivery of active pharmaceutical ingredients such as phytochemicals,
vaccines, and antineoplastic agents to deeper layers of the skin. The primary factor for the
transfer of transfersomes into the inner epidermal layers is the osmotic gradient.
Transferosmes are administered under nonocclusive conditions to make sure the transport is
supported my skin's innate hydration gradient. This review outlines the evolution of
transferosomes, it's components and the dermal kinetics. . Commons License Researchers
have found that these delivery systems are one of the successful ways to deliver wide range
drugs, peptides, proteins and nucleic acids. They have a potential to carry large molecules
which were previously limited with other systems. Furthermore, it has a promising future as a
nanocarrier system integrated with the computational method saves the time and effort to get
the best possible formulation composition. Despite many advantages and applications,
transferosome still have few limitations which need to be addressed. Scaling up process of
transferosomes, delivery of lipophilic drugs and leaching out of hydrophilic molecules are
some of the issues which have been reported often. Additionally, limited clinical trials
completed, suggests the urgent need to conduct more clinical studies to provide safety and
efficacy profile for transfersomes. Additional research on transfersomes may result in
innovative, potential therapeutic strategies against a variety of disorders.

27
 [Link]

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