Field of Invention
The present invention is directed to a process for preparing liposomes, and the
preparation of liposomes suitable for cosmetics and dermatological applications.
Background and Related Art
Liposomes are small, spherical vesicles composed primarily of various types of lipids,
phospholipids and secondary lipophilic components. Usually, these elements are
arranged in a bilayer formation, similar to the lipid arrangement of biological
membranes. Typically, the polar end of a component lipid or lipid-like molecule is in
contact with the surrounding solution, usually aqueous solution, while the non-polar,
hydrophobic end of the lipid or lipid-like molecule is in contact with the non-polar,
hydrophobic end of another lipid or lipid-like molecule. The resulting bilayer membrane
is selectively permeable to molecules of a certain size, hydrophobicity, shape, and net
charge.
The lipid bilayer of liposomes mimics the structure of cell membrane thereby being one
of the highly biocompatible delivery system. They are used to deliver molecules which
are not easily soluble in water, or when a directed, timed release is desired. Because of
their selective permeability to many chemical compounds, liposomes are useful as
delivery vehicles for biological materials and compounds that are hydrophobic and
hydrophilic in nature. They provide several advantages in the delivery of active
ingredients. They find many applications including therapeutic, diagnostic and
cosmetic. In the recent years, they find a very important role, particularly, in cosmetics
and dermatological applications. The compound(s) that are to be delivered can be
provided within the liposomes (hydrophilic active ingredients in the aqueous core),
where they remain protected from the outside environment thus enhancing the stability
of the active ingredients. Alternatively, the compounds that are targeted for delivery can
be incorporated into the lipid bilayer of the liposomes if they are lipophilic or have been
chemically linked to the lipids. Upon reaching the target site, the liposomes may be

degraded or they may fuse with the membranes of cells resulting in release of the
compound(s).
Several methods of preparing liposomes are known however these methods typically
employ solvents such as methylene chloride, chloroform, methonal, hexane, ether and
the like, which are hazardous.
US 4089801 describes a process for preparation liposomes, which involves dispersing a
first aqueous liquid with the aid of ultrasonic vibration in a water-immiscible carrier
liquid less denser than water. The process comprises steps of (a) dispersing a first
aqueous liquid consisting of a solution of a biologically active substance, with the aid of
ultrasonic vibration in a carrier liquid which is insoluble in water or only slightly
soluble in water and has a density less than that of water, in the presence of at least one
interface compound (lipids); (b) combining this dispersion with a second aqueous liquid
to form a heterogeneous two-layer system comprising an upper liquid layer formed by
the dispersion and a lower liquid layer formed by the second aqueous liquid, the two
layers being separated by the monomolecular film of the said interface compound; and
(c) centrifuging the two layer system at an angular velocity of about 30,000 rev/min to
force the dispersed globules of the first aqueous liquid by gravity through the
monomolecular separating film of the interface compound and into the lower layer of
the second aqueous liquid.
US 4933121 describes a method of preparation of liposomes that maintains a
temperature below the phase transition temperature of the lipid used to make the
liposome both during the hydration of the lipid film and, if used, during an extrusion
step. The preparation method comprises the steps of a) lipid film preparation; b) lipid
film hydration; c) liposome dialysis; d) liposome extrusion; and e) liposome washing.
US 4308166 describes an extemporaneous preparation method of liposomes, which
involves incorporating englobulated therapeutically active substances characterized in
that an aspirated phospholipid emulsion in water or physiologic solution is introduced
into a container containing the active substance as a dry powder or lyophilate. The

method consists in using the technique normally adopted for dissolving a dry powder or
lyophilate with a solvent, that is: a)aspirate the phospholipid emulsion with a syringe; b)
discharge the contents of the syringe into a container (vial or bottle) of the
therapeutically active substance, in the dry or lyophilized state; c) agitate the vial or
bottle to improve the solubility; d) aspirate the solution with the syringe, eventually
discharging it again into the container to aspirate it anew. At this point, the syringe
contains a fine suspension of liposomes in an aqueous environment which englobe
within their structure the therapeutically active substance.
US 4731210 describes a method of preparation of liposomes using associates of at least
one amphiphilic bilayer-forming substance and a solubilizing agent are formed in an
aqueous phase.
Further JT Buboltz et. al. in "A novel strategy for the preparation of liposomes: rapid
solvent exchange"; BiochimBiophysActa. 1999 1417(2):232-45. - Elsevier describes a
process for preparation of liposomes using rapid solvent exchange, http://ac.els-
cdn.com/S0005273699000061/l-s2.0-S0005273699000061-main.pdf?_tid=6f9aad2a-
f229-lle2-b92e-
00000aacb360&acdnat=1374427235_189c6bl7f678b70705c56851abd007fb
http://www.sciencedirect.com/science/article/pii/S0005273699000061
F Szoka Jr, D Papahadjopoulos in "Procedure for preparation of liposomes with large
internal aqueous space and high capture by reverse-phase evaporation -
ProcNatlAcadSci USA. 1978 September; 75(9): 4194-4198 describes a process for
preparation of liposomes with large internal aqueous space and high capture by reverse-
phase evaporation.http://www.pnas.org/content/75/9/4194.full.pdf
Heurtault et. al in "A novel phase inversion-based process for the preparation of lipid
nanocarriers - June 2002, Volume 19, Issue 6, pp 875-880 describes a process for
preparation of liposomes using phase inversion techniques.
http://link.springer.eom/article/10.1023%2FA%3A1016121319668#page-l.

Besides employing organic solvents which are hazardous in nature, the prior art
methods of preparation are complex and cumbersome, involving multiple steps thus
failing to establish a commercially effective preparation method. Moreover, it is
difficult to adapt such processes in the industrial set up during large scale production
process. These processes also involve energy intensive techniques such as high pressure
homogenization, ultrasound, that are difficult to scale-up. Therefore there exists a need
to develop a simple, industry scalable process for the preparation of liposomes.
Further, most of such processes involve use of alcohol. Use of alcohol in industry is not
desirable as it is not environmental friendly therefore there exist need to develop an
alcohol free process for the preparation of liposomes.
Some of these methods involve dissolution of lipids in carrier liquids (organic solvents)
or disperse lipids using emulsifiers followed by removal of organic solvents from the
system. The removal process under reduced pressure or otherwise, at times, fails to
remove the solvent completely. Yet again, the processes disclosed in the prior art
require heating for liposome formation.
Therefore, there exists a need to develop a method, which is simple, not cumbersome
and commercially viable for preparing liposomes.
Accordingly, present invention provides a simple, convenient and cost-efficient process
for preparing liposomes.
Objects of the Invention
It is an object of the present invention to overcome the disadvantages of the prior arts.
It is another object of the present invention is to provide a simple, convenient and cost
efficient process for preparation of liposomes.

It is yet another object of the present invention is to provide a process for preparation of
liposomes that can be easily scaled up.
It is another object of the present invention is to provide a process for preparation of
liposomes in a single step.
It is yet another object of the present invention is to provide a process for preparation of
liposomes that avoids use of organic solvents such as methylene chloride, chloroform,
methanol, hexane, ether and the like which are hazardous in nature.
It is yet another object of the present invention is to provide a process for preparation of
liposomes that has applications in the pharmaceutical, cosmetic and dermatological
field.
SUMMARY OF THE INVENTION
In one aspect the present invention provides a process for the preparation of liposomes
comprising homogenization of a mixture of:
a) water; and
b) a preformed solution of
i) at least one phospholipid; and
ii) at least one water soluble solvent;
wherein at least one phospholipid is phosphatidylcholine and homogenization is
performed at about 3000 rpm to about 20000 rpm and the amount of lipid in the water-
soluble solvents ranges from 0.001 to 30% wt/wt percentage.
In one other aspect the present invention provides a process for preparation of
liposomes that is simple, convenient, cost efficient and avoids use of surfactants and / or
short chain alcohols.

In another aspect the present invention provides a process for preparation of liposomes
that obviates use of hazardous organic solvents such as methylene chloride, chloroform
and the like.
In yet another aspect the present invention provides a single step process for preparation
of liposomes that can be easily scaled up to large scale industry batch preparation.
In another aspect the invention provides personal care compositions comprising
liposomes formed by the process disclosed herein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process for preparation of liposomes that involves a
single step and can be easily scaled up to large-scale industrial batch production.
Liposomes are typically amphipathic materials which can form a closed lamellar bilayer
phase, plus additional materials to be delivered or useful in targeting delivery or
conferring useful properties on the liposome such as extended half-life, and solvent.
Mixtures of components may be used. Lipids and lipid-like components suitable for use
in this invention include lipids, phospholipid, a mixture of phospholipid, and their
derivatives.
Suitable phospholipids include Phosphatidylcholine (lecithin) (PC), phosphatidyl
ethanolamine (PE), phosphatidyl serine (PS) lyso phospholipids, Phosphatidic acid
(phosphatidate) (PA) , Phosphoinositides such as Phosphatidylinositol (PI),
Phosphatidylinositol phosphate (PIP), Phosphatidylinositol bisphosphate (PIP2),
Phosphatidylinositol triphosphate (PIP3), Phosphosphingolipids such as
Ceramidephosphorylcholine (Sphingomyelin) (SPH), Ceramidephosphorylethanolamine
(Sphingomyelin) (Cer-PE), Ceramidephosphoryllipid, natural phospholipids such as egg
PC, egg PG, soy PC, hydrogenated soy PC, sphingomyelin, lecithin separated from soya
lecithin, egg lecithin, rice bran lecithin, combinations and derivatives thereof.

Suitable water-soluble solvents include propylene glycol, butylene glycol, poly ethylene
glycol, sorbitol, glycerine and mixture thereof.
In one embodiment of the present invention the process for the preparation of liposomes
comprising homogenization of a mixture of:
a) water; and
b) a preformed solution of
i) at least one phospholipid; and
ii) at least one water soluble solvent;
wherein at least one phospholipid is phosphatidylcholine and homogenization is
performed at about 3000 rpm to about 20000 rpm and the amount of lipid in the water-
soluble solvents ranges from 0.001 to 30% wt/wt percentage.
In another embodiment of the present invention the phospholipid may be present at a
concentration of from about 0.1 wt% to about 30 wt%, the water soluble solvent may be
present at a concentration of from about 0.1 wt% to about 30 wt% and water may be
present at upto70% wt/wt concentration. The system assumes a gel-like appearance,
when the amount of lipid and solvent exceeds some limits (>30%), instead of forming
liposomal system.
The liposome preparation process of the current invention is not temperature sensitive
and essentially does not require high temperatures for its operation.
Moreover the process of the present invention does not employ cost intensive
techniques like high pressure homogenization, ultrasonication, extruder sonication.
Besides being cost intensive, it is very difficult to adapt these techniques for industrial
preparation of liposome. It is known that lab scale high pressure homogenizers cost
more than Rupees 25-50 lakhs, while the simple lab scale homogenizer as employed in
the present invention cost only about rupees 5-10 lakhs. Therefore, the cost reduction is
almost five times.

In another embodiment of the present invention, homogenization is performed at
ambient temperature. Ambient temperature is room temperature ranging from 15 to
40°C. Since the present invention involves simple dispersion, the industrial production
can be done by simply using a homogenizer that rotates at 3000 to 20000 rpm. The
amount of lipid in the water-soluble solvents varies from 0.001 to 30% wt/wt
percentage.
In yet another embodiment of the present invention, personal care compositions
comprising liposomes of the present invention along with other adjuvants and active
agents may be prepared.
In one preferred embodiment the liposome composition formed by the present invention
may further comprise oil soluble actives selected from a group comprising but not
limited to skin lightening agents, darkening agents such as self-tanning agents, anti-acne
agents, shine control agents, anti-microbial agents, anti-inflammatory agents, anti-aging
agents, in particular anti-wrinkle agents, anti-mycotic agents, anti-parasite agents,
external analgesics, sunscreens, photoprotectors, antioxidants, keratolytic agents,
detergents/surfactants, moisturizers, nutrients, vitamins, energy enhancers, anti-
perspiration agents, astringents, deodorants, hair removers, firming agents, anti-callous
agents, and agents for hair, nail, and/or skin conditioning. These oil soluble actives may
be added to the preformed solution of phospholipid (s) and water soluble solvent (s)
before it is added to water and homogenized. The oil soluble actives may be present in
the liposome composition at a concentration of from about 0.001 wt% to about 25 wt%
In another preferred embodiment the liposome composition formed by the present
invention may further comprise water-soluble active. Such water soluble actives include
skin lightening agents, darkening agents such as self-tanning agents, anti-acne agents,
shine control agents, anti-microbial agents, anti-inflammatory agents, anti-aging agents,
in particular anti-wrinkle agents, anti-mycotic agents, anti-parasite agents, external
analgesics, sunscreens, photoprotectors, antioxidants, keratolytic agents,

detergents/surfactants, moisturizers, nutrients, vitamins, energy enhancers, anti-
perspiration agents, astringents, deodorants, hair removers, firming agents, anti-callous
agents, and agents for hair, nail, and/or skin conditioning. These water-soluble active(s)
actives may be added to water before the preformed solution of phospholipid (s) and
water soluble solvent is added to it and homogenized. The water soluble actives may be
present in the liposome composition at a concentration of from about 0.001 wt% to
about 25 wt%.
In another preferred embodiment the present invention is used for the large scale
preparation of liposomes. Liposomes have wide usage in the cosmetic industry to
deliver actives to the skin. Some of the products that can be processed in large scale in
industry are under eye serum, local delivery formulation, leave on products, anti ageing
cream, anti-acne, skin lightening, anti blemish and anti-cellulite formulation.
In yet another preferred embodiment the present invention may also contain adjuvants
which are conventional in the cosmetic or dermatological field, such as hydrophilic or
lipophilic gelling agents, preservatives, antioxidants, solvents, fragrances, fillers,
opacifiers, bactericides, odor absorbers and dyestuffs or colorants. Depending on their
nature, these additives and adjuvants may be introduced into the fatty phase or into the
aqueous phase.
Advantage of the present process:
1. It can be processed at ambient temperature and hence suitable for incorporating
both oil-soluble and water-soluble temperature sensitive actives.
2. It can encapsulate both water soluble (hydrophilic) and oil soluble actives
(hydrophobic).
3. It does not employs use of alcohol and hence make it environmental friendly.
4. It reduces time and cost of production

5. Since simple dispersion is required, the industrial production can be done by
simply using a homogenizer that rotates at 3000 to 20000 rpm
EXAMPLES
Non limiting examples of the process of the present invention
The process for the preparation of liposomes containing water-soluble and oil-soluble
active/s is prepared in accordance to the below exemplified process.
Preparation of the preformed solution:
Example 1
Step 1: Preparing phospholipid solution in a water soluble solvent by heating it at 60-70
°C by gentle mixing for 60-120 min and cooling the same at ambient temperature.
Step 2. Weigh required amount of lipid solution as obtained in step 1 into a container at
ambient temperature
Step 3. Weigh oil-soluble active and add the same in the lipid solution as mentioned in
step 2 and mix well under stirring to get a homogeneous solution at ambient temperature
Step 4. Weigh required amount of water-soluble active/s ingredient/s in a container.
Step 5. Weigh required amount of water and add the same in the container as mentioned
in step 4 and mix under stirring to get a homogeneous solution
Process for preparing the liposome:
Weigh required amount of lipid solution as obtained in step 3 and disperse the same into
the mixture as mentioned in step 5 at an ambient temperature under homogenization at
3000 to 20000 rpm until a transparent/translucent system is formed.

The amount of lipid in the water-soluble solvents varies from 0.001 to 30% wt/wt
percentage.
It was observed that the system assumes a gel-like appearance, when the amount of lipid
and solvent exceeds some limits (>30% wt/wt), instead of forming liposomal system.
Example 2:
Step 1: weigh 50 gm of phospholipids in a 250 ml container.
Step 2: Add 50 gm of propylene glycol to the container of step 1.
Step 3: Heat the mixture of step 2 to around 60-70 °C with gentle stirring until a
homogenous solution is obtained.
Step 4: Cool the solution as obtained in step 3 at ambient temperature.
Step 5: Weigh 100 gm of water in a 250 ml container.
Step 6: Add 20 gm mixture of step 4 in the solution of step 5 under homogenization at
3,000 to 20,000 rpm for 5-20 minutes until a transparent/translucent system is obtained.
Example 3:
Step 1: Weigh 25 gm of propylene glycol and 25 gm of glycerol in a 250 ml container
and mix well.
Step 2: Weigh 50 gm of phospholipids and add to the mixture of step 1 under gentle
stirring.
Step 3: Heat the mixture of step 2 to around 60-70°C with gentle stirring until a
homogenous solution is obtained.
Step 4: Cool the solution as obtained in step 3 at ambient temperature.
Step 5: Weigh 100 gm of water in a 250 ml container.
Step 6: Add 20 gm mixture of step 4 in the solution of step 5 under homogenization at
3,000 to 20,000 rpm for 5-20 minutes until a transparent/translucent system is obtained.

Example 4
Step 1: weigh 50 gm of phospholipids in a 250 ml container.
Step 2: Add 50 gm of propylene glycol to the container of step 1.
Step 3: Heat the mixture of step 2 to around 60-70 °C with gentle stirring until a
homogenous solution is obtained.
Step 4: Cool the solution as obtained in step 3 at ambient temperature.
Step 5: Weigh 50 gm of water in a 250 ml container.
Step 6: Add 100 gm mixture of step 4 in the solution of step 5 under homogenization at
3,000 to 20,000 rpm for 5-20 minutes
Observation: The amount of lipid in solvent was 33.3% by weight. The solution
obtained was not the desired transparent/translucent system instead a highly viscous gel-
like structure was obtained.
Example 5
A litre of liposomes is prepared according to the processes exemplified in examples 1
and 2 is 10 minutes.
Thus, considering increased volume of 100 It, preparation time would be about 40 - 45
minutes.
For the same amount (1 lit) of liposome prepared by conventional processes employ
high pressure homogenization, passing the lipid dispersion through a high pressure
system for at least 10 cycles to get a homogenous system. Each cycle takes around 10
minutes. Thus, liposome preparation using high pressure homogenization takes 1-2
hours/It. Considering the increased volume of 100 litres liposome preparation using
high pressure homogenization will take at least 5 hours.
Example 6

Process employing a high pressure homogenizer
1. Dissolve 50 g of lipid in 50 ml chloroform in a 250 ml container and evaporate the
chloroform using rotary evaporator to make a thin film of the lipid for 3-5 hrs until the
last trace of chloroform was removed from the lipid layer.
2. Add 100 gm water in the container and hydrate the dried lipid layer for 1 - 2 hours for
swelling.
3. Stir the swelled lipid in buffer to make a lipid dispersion (multilamellar vesicles).
4. Pass the lipid dispersion through a high pressure homogenizer for at least 10-15
cycles for 30 minutes -1 hour to obtain liposomes (unilamellar liposomes).
Hence the total time taken for completing the liposomal preparation process is 4 - 7
hours, while the process of the present invention 40 to 45 minutes for 100 litres of
liposomes.
Example 7


1. Make a solution of lipid in propylene glycol and add vitamin E acetate to it.
2. Mix glycerine, vitamin C derivative, peptide, sodium hyaluronate, d- Panthenol
and initiate homogenization of the same at about 4000 RPM.
3. Slowly add the solution of step 1 in the solution of step 2 during homogenization
and continue homogenization for 10 minutes to obtain liposomal solution.
4. Finally add DMDM hydantoin to the liposomal solution of step 3 and mix well.

WE CLAIM:
1. A process for the preparation of liposomes comprising homogenization of a mixture
of:
a) water; and
b) a preformed solution of
i) at least one phospholipid; and
ii) at least one water soluble solvent;
wherein homogenization is performed at about 3000 rpm to about 20000 rpm
and the amount of lipid in the water-soluble solvents ranges from 0.001 to 30%
wt/wt.
2. The process as claimed in claim 1, wherein the amount of water soluble solvent
ranges from 0.1 wt% to 30 wt%.
3. The process as claimed in claim 1, wherein the phospholipid is selected from the
group consisting of Phosphatidylcholine (lecithin) (PC),phosphatidyl
ethanolamine (PE), phosphatidyl serine (PS) lyso phospholipids, Phosphatidic
acid (phosphatidate) (PA) , Phosphoinositides such as Phosphatidylinositol (PI),
Phosphatidylinositol phosphate (PIP), Phosphatidylinositol bisphosphate (PIP2),
Phosphatidylinositol triphosphate (PIP3), Phosphosphingolipids such as
Ceramidephosphorylcholine (Sphingomyelin) (SPH),
Ceramidephosphorylethanolamine (Sphingomyelin) (Cer-PE),
Ceramidephosphoryllipid, natural phospholipids such as egg PC, egg PG, soy
PC, hydrogenated soy PC, sphingomyelin, lecithin separated from soya lecithin,
egg lecithin, rice bran lecithin, combinations and derivatives thereof.
4. The process as claimed in claim 3, wherein at least one phospholipid is
phosphatidylcholine.
5. The process as claimed in claim 1, wherein the water soluble solvent is selected
from propylene glycol, butylene glycol, poly ethylene glycol, sorbitol, glycerine
and mixture thereof.
6. The process as claimed in claim 1, wherein homogenization is performed at
temperature between 15°C to 40°C.

7. The process as claimed in claim 1, wherein homogenization is performed for 5
to 20 minutes.
8. A personal care composition comprising liposome as prepared by the process
claimed in claim 1, adjuvants, and active agents.
9. The composition as claimed in claim 8, wherein the active agents can be selected
from a group comprising lightening agents, darkening agents such as self-
tanning agents, anti-acne agents, shine control agents, anti-microbial agents,
anti-inflammatory agents, anti-aging agents, in particular anti-wrinkle agents,
anti-mycotic agents, anti-parasite agents, external analgesics, sunscreens,
photoprotectors, antioxidants, keratolytic agents, detergents/surfactants,
moisturizers, nutrients, vitamins, energy enhancers, anti-perspiration agents,
astringents, deodorants, hair removers, firming agents, anti-callous agents, and
agents for hair, nail, and/or skin conditioning.
10. The composition as claimed in claim 8, wherein the adjuvant can be selected
from a group comprising hydrophilic or lipophilic gelling agents, preservatives,
antioxidants, solvents, fragrances, fillers, opacifiers, bactericides, odor absorbers
and dyestuffs and colorants.

ABSTRACT

A process for the preparation of liposomes comprising homogenization of a mixture of
water and a preformed solution of (i) at least one phospholipid; and (ii) at least one
water soluble solvent; wherein at least one phospholipid is phosphatidylcholine and
homogenization is performed at about 3000 rpm to about 20000 rpm and the amount of
lipid in the water-soluble solvents ranges from 0.001 to 30% wt/wt percentage.