ISSN: 0975 -766X CODEN: IJPTFI Available Online through ... · PDF fileNovel drug delivery...

Rabiya Ansari*et al. /International Journal of Pharmacy & Technology

IJPT| June-2017| Vol. 9 | Issue No.2 | 29735-29758 Page 29735

ISSN: 0975-766X

CODEN: IJPTFI

Available Online through Research Article

www.ijptonline.com LIPOSOMES AS A NOVEL DRUG DELIVERY SYSTEM

Rabiya Ansari*, Abdul Mannan

Department of Pharmaceutics, Deccan School of Pharmacy, Hyderabad, Telangana 500001.

Email: [email protected]

Received on: 25-02-2017 Accepted on: 28-03-2017

Abstract

This review article is intended to provide an overview of liposomes as a novel drug delivery system. It has focused on

the structure, components, mechanism of liposome formation, advantages, disadvantages, classification, method of

preparation, characterization, stability and applications of the liposomes Liposomes are spherical shaped bilayered

vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer. The hydration of a dry lipid

film lead to the formation of liposome. Liposomes have a wide range of applications in science, pharmaceutical

industry, as a drug carrier, anti cancer agent and in dermatology and cosmetics. Liposome as novel drug delivery

systems has played a significant role in formulation of potent drug to improve therapeutics.

Keywords: Liposome, Vesicles, Amphiphilic, Hydrophilic, Hydrophobic, Phospholipids, Novel Drug Delivery

System.

Introduction

Novel drug delivery system aims at providing some control, whether this is of time related or dimensional nature, or

both, of drug release in the body. Novel drug delivery attempts to either sustain drug action at a predetermined rate,

or by maintaining a relatively constant, effective drug level in the body associated with minimization of undesirable

side effects [1]

. Different types of pharmaceutical carriers are present. They are – particulate, polymeric, and

macromolecular. Particulate type carrier also known as colloidal carrier system, which includes lipid particles i.e. low

and high density lipoprotein- LDL and HDL respectively, microspheres, nanoparticles, polymeric micelles and the

vesicular like liposomes, niosomespharmacosomes, virosomes, etc[2]

. Liposomes are simple microscopic vesicles.

Structurally, liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a

membranous lipid bilayer mainly composed of natural or synthetic phospholipid [3]

. Generally, liposomes are

spherical vesicles with particle sizes ranging from 50-150 nm to several micrometers [4]

.

http://www.ijptonline.com/



Bangham and colleagues first discovered liposomes in early 1960’s. They were discovered when Bangham and R. W.

Horne. They were testing the institute's new electron microscope by adding negative stain to dry phospholipids. The

resemblance to the plasma lemma was obvious, and the microscope pictures served as the first real evidence for the

cell membrane being a bilayer lipid structures[5,6]

.

The name liposome is derived from two Greek words ‘Lipid’ meaning fat and ‘Soma’ meaning body and it

subsequently became the new most extensive explore drug delivery system. The hydration of a dry lipid film was

found to lead to the formation of enclosed spherical vesicles or liposomes that resemble miniature cellular organelles

with lipid bilayers [7]

. A free drug injected in bloodstream typically achieves therapeutic level for short duration due

to metabolism and excretion. Drug encapsulated by liposome achieve therapeutic level for long duration as, drug

must first be release from liposome before metabolism and excretion [8]

. Liposome drug delivery is gaining interest

due to its contribution to varied areas like drug delivery, cosmetics, and structure of biological membrane [9]

.

Structure and Components of Liposomes

Liposomes are colloidal particles. There are number of components of liposomes, however phospholipids and

cholesterol are the main components.

Most liposome formulations approved for human use contain phosphotidyl choline with a fraction of cholesterol is

often included in it.

Phospholipids are the major structural components of biological membranes. The most common phospholipid is

phosphatidylcholine (PC) molecule. It is an amphiphatic molecule in which a glycerol bridge links a pair of

hydrophobic acyl hydrocarbon chains with a hydrophilic polar head group phosphocholine[10]

.

Examples of phospholipids are- Phosphatidyl choline (Lecithin) – PC, Phosphatidyl ethanolamine(cephalin) –PE,

Phosphatidyl serine – PS, Phosphatidyl inositol – PI, Phosphatidyl Glycerol – PG.

Figure 1: Some Common Naturally Occuring Phosphatidyl Phospholipids [7]

.



The general chemical structure of phospholipids shows a glyceral backbone. At a position of number 3 of the glyceral

molecule, the hydroxyl group is esterified to phosphoric acid. The hydroxyl groups at positions 1 and 2 of the

glycerol are usually esterified with long chain fatty acids. One of the remaining oxygen groups of the phosphoric acid

may be further esterified to a variety of organic molecules including glycerol, choline, ethanolamine, serine and

inositol [7].

Sphingolipids backbone is sphingosine or a related base. Most common Sphingolipids are Sphingomyelin,

Glycosphingo lipids. These are included in liposomes to provide a layer of surface charged group [2,7].

Cholesterol has a steroid backbone, and its derivatives are included in liposome preparation to improve their bilayer

characteristics, rigidity of the bilayer membrane, reduces the permeability of water-soluble molecules through the

membrane and improves the stability of the bilayer membrane in the presence of biological fluids such as blood and

plasma. The cholesterol molecule orients itself among the phospholipid molecules with its hydroxyl group facing

towards the water phase and the tricyclic ring sandwiched between the first few carbons of fatty acyl chains into the

hydrocarbon core of the bilayer [7,10].

Figure 2: Structure of Liposome [11,12].

Figure 3: Structure of Liposome [11,12].

Advantages

Can be administered through various routes [1].

Provides selective passive targeting to tumor tissues, example, liposomal doxorubicin [4].



Alter pharmacokinetics and pharmacodynamics of drugs.

Suitable for delivery of hydrophobic, amphipathic and hydrophilic drugs [13].

Protect the encapsulated drug from the external environment [14].

Reduced toxicity and increased stability [15].

Reduce exposure of sensitive tissues to toxic drugs [16].

Provide sustained release.

Direct interaction of the drug with cell biodegradable and flexible [17].

Disadvantages

Allergic reactions may occur to liposomal constituents [1].

Low solubility [2].

Liposomes are extensively used as carriers for molecules in cosmetic and pharmaceutical industries.

Short half life [18].

Leakage and fusion [19].

Less stability.

Phospholipids undergoes oxidation, hydrolysis.

High production cost [20].

Liposomes can trap both hydrophobic and hydrophilic compounds, avoid decomposition of the entrapped

combinations, and release the entrapped at designated targets [21].

Classification of Liposomes

Liposomes are classified on the basis of Structure, Method of preparation, Composition and application,

Convectional liposome and Specialty liposome [2,11,16,18,19,20,22,23].

Figure 4: Classification of Liposomes.

CLASSIFICATION OF LIPOSOMES

Structure Method of Preparation

Composition and Application

Convectional Liposome

Specialty Liposome



Classification Based on Structure (Table 1)

Table 1- Vesicle Types with their Size and Number of Lipid Layers [23]

Based on Method of Preparation (Table 2)

Table 2- Different Preparation Methods and the Vesicles Formed by these Methods [23]

PREPARATION METHOD VESICLE TYPE

Single or oligo lamellar vesicle made by reverse phase evaporation

method

REV

Multi lamellar vesicle made by reverse phase evaporation method MLV-REV

Stable plurilamellar vesicle SPLV

Frozen and thawed multi lamellar vesicles FATMLV

Vesicle prepared by extrusion technique VET

Dehydration-rehydration method DRV

VESICLE TYPE ABBREVIATION DIAMETER SIZE NUMBER OF LIPID

BILAYER

Unilamellar vesicle UV All size range One

Small unilamellar vesicle SUV 20-100 nm One

Medium unilamellar

vesicle

MUV More than 100nm One

Large unilamellar vesicle LUV More than 100 nm One

Giant unilamellar vesicle GUV More than one micro

meter

One

Oligolamellar vesicle OLV 0.1-1 micrometer Approximately 5

Multilamellar vesicle MLV More than 0.5 5-25

Multi vesicular vesicle MV More than one micro

meter

Multi compartment

structure



Based on Composition and Application (Table 3)

Table 3- Different Liposome with their Compositions [23]

Based Upon Conventional Liposome

Stabilize natural lecithin (PC) mixtures

Synthetic identical, chain phospholipids

Glycolipids containing liposomes

Based Upon Specialty Liposome

Bipolar fatty acid

Antibody directed liposome

Methyl/Methylene x-linked liposome

Lipoprotein coated liposome

Carbohydrate coated liposome

Multiple encapsulated liposome

Mechanism of Formation of Liposomes

The basic part of liposome is formed by phospholipids, which are amphiphilic molecules having a hydrophilic head

and hydrophobic tail. The hydrophilic part is mainly phosphoric acid bound to a water soluble molecule, whereas, the

hydrophobic part consists of two fatty acid chains with 10 – 24 carbon atoms and 0 – 6 double bonds in each chain.

When these phospholipids are dispersed in aqueous medium, they form lamellar sheets by organizing in such a way

TYPE OF

LIPOSOME

ABBREVIATION COMPOSITION

Conventional

liposome

CL Neutral or negatively charge

phospholipid and cholesterol

Fusogenic

liposome

RSVE Reconstituted sendai virus envelops

PH sensitive

liposome

- Phospholipid such as PER or DOPE

with either CHEMS or OA

Cationic liposome - Cationic lipid with DOPE

Long circulatory

liposome

LCL Neutral high temperature, cholesterol,

and 5-10% PEG, DSP

Immunolatory

liposome

IL CL or LCL with attached monoclonal

antibody or recognition sequences



that, the polar head group faces outwards to the aqueous region while the fatty acid groups face each other and finally

form spherical vesicle like structures called as liposomes. The polar portion remains in contact with aqueous region

along with shielding of the non-polar part, which is oriented at an angle to the membrane surface [24]. It is the

hydrophilic/ hydrophobic interactions between lipid – lipid, lipid – water molecules that lead to the formation of

bilayered vesicles in order to achieve a thermodynamic equilibrium in the aqueous phase [25].

Figure 5: Mechanism of Formation of Liposomes [4]

Methods of Liposome Preparation

Liposomes can be prepared by active loading or passive loading techniques [4,26,27,28].

Figure 6: Methods Of Preparation Of Liposomes [4]



I) PASSIVE LOADING TECHNIQUES

A) MECHANICAL DISPERSION METHODS

Figure 7: Preparation of Liposomes through Mechanical Dispersion Method.

Thin Film Hydration Using Hand Shaking (MLVS) And Non-Hand Shaking Methods (LUVS)

Lipids are casted as stacks of film from their organic solution using flash rotary evaporator under reduced pressure or

by hand shaking and then the casted film is dispersed in an aqueous medium. Upon hydration the lipids swell and

peel off from the wall of the round bottom flask and vesiculate forming multi lamellar vesicles (MLVs). The

mechanical energy required for the swelling of lipids and dispersion of casted lipid film is imparted by manual

agitation or hand shaking technique or by exposing the film to the stream of water-saturated nitrogen for 15mins

followed by swelling in aqueous medium without shaking. Hand shaking method produces MLVs whereas non-

handshaking method produces LUVs. The percent encapsulation efficiency as high as 30% is achieved [4].

Figure 8: Preparation of Liposomes by Thin Film Hydration Using Hand Shaking (MLVS) and Non-Hand

Shaking Methods (LUVS) [4]

liposomes

lipids swell and hydrate

hydrated using aqueous buffer

solid lipid mixture

organic solvent removed by film deposition under vacuum

lipid co-dissolved in organic solvent



Micro Emulsification

Micro fluidizer is used to prepare small MLVs. The lipids can be introduced into the fluidizer, either as a dispersion

of large MLVs or as a slurry of unhydrated lipid in an organic medium. It pumps the fluid at very high pressure about

10,000 psi or 600-700 bar through a 5 µm orifice. Then it is forced along defined micro channels, which direct two

streams of fluid to collide together at right angles at a very high velocity. The fluid collected can be recycled through

the pump and interaction chamber until vesicles of spherical dimensions are obtained. After a single pass, the size of

diameter is reduced to 0.1 and 0.2 µm in diameter. This method has the advantage of being able to process samples

with a very high proportion of lipids i.e. 20% or more by weight. This process is efficient for encapsulation of water

soluble materials. Percentage capture values upto 70% have been reported, starting with lipid concentration of

approximately 200mg/ml [4].

Figure 9: Preparation of Liposomes by Micro Emulsification [4]

Sonication

Sonication is the most extensively used method for the preparation of SUV. The main disadvantages of this method

are very low encapsulation efficacy and presence of MLV along with SUV. There are two sonication techniques i.e.

probe or bath sonicator. The probe is employed for dispersions which require high concentration of lipids while bath

sonicator is more suitable for large volumes of diluted lipids. Probe tip sonicator tends to release titanium particles

into the liposome dispersion; because of this bath sonicator is most widely used. Sonication of MLV dispersion is

done by placing a test tube containing the dispersion in the bath sonicator and sonicating it for 5-10minutes. Above

Tc of the constituent lipid the lipid dispersion begins to clarify to yield a slightly hazy transparent solution.

Centrifugation is done to yield a clear SUV dispersion and then the dispersion is placed in a clear plastic walled

ultracentrifuge tube. The dispersion is generally centrifuged at 100,000g, 30minutes, 20 degree Celsius to sediment

titanium particles and large MLVs followed by higher speed centrifugation. i.e. 1,59,000g for 3-4 hours. After



spinning, the tube is carefully removed from the rotor and with the help of Pasteur pipette, the liquid with top clear

layer is decanted leaving the central opalescent layer containing small MLVs and a pellet behind. The top layer

constitutes pure dispersion of SUVs. [4].

Figure 10: Preparation of Liposomes by Sonication [4]

French Pressure Cell Method

Here extrusion of large liposomes in a French press at a very high pressure is performed. This technique yields

unilamellar or oligolamellar liposomes of intermediate size 30-80 nm in diameter depending on the applied pressure.

The sizes of resulting French press extruded liposomes are variable, depending upon the lipid composition, the

temperature and most important on pressure. The disadvantage of this method includes high initial cost of the press

that consists of hydraulic press and pressure cell. The advantage of this method is the liposomes prepared by this

method are less likely to suffer from the structural defects and instabilities [4].

Figure 11: Preparation of Liposomes by French Pressure Cell Method [4]

Membrane Extrusion Method

Here, the size of liposomes is reduced by gently passing them through membrane filter or defined pore size at much

lower pressure i.e. less than 100 psi. The membrane extrusion technique can be used to process LUVS as well as

MLVs. In this process, the vesicle contents are exchanged with the dispersion medium during breaking and resealing



of phospholipid bilayers as they pass through the polycarbonate membrane. The liposomes produced by this

technique are termed as LUVETs. The 30% capture volume can be obtained using high lipid concentration i.e.

300mMPC. The trapped volume in this process is 1-2 litre/mol of lipids[4].

Figure 12: Preparation of Liposomes by Membrane Extrusion Method [4]

Dried Reconstituted Vesicles

Liposomes obtained by this method are usually unilamellar or oligolamellar of the order of 1µm or less in diameter.

The first step of freeze drying is used to freeze and lyophilize a performed SUVs dispersion. This leads to an

organized membrane structure which on addition of water can rehydrate, fuse and reseal to form vesicles with a high

capture efficiency. The water soluble matrix to be entrapped are added to the dispersion of empty SUVs and they are

dried together. The advantages of this method include high entrapment of water soluble component and the use of

mild conditions for the preparation and loading of bioactives. The disadvantage is this method is suitable only for

unilamellar vesicles i.e. SUVs [4].

Figure 13: Preparation of Liposomes by Dried Reconstituted Vesicles and Freeze Thaw Method [20]

Freeze Thaw Sonication



This method is an extension of dried reconstituted vesicles. Here, SUVs are rapidly freezed followed by slow thawing

by standing at room temperature for 15minutes and finally subjecting it to sonication cycle which disperses aggre-

gated materials to LUV. The formation of unilamellar vesicles is due to the fusion of SUV. The encapsulation

efficiencies from 20–30% were obtained. The disadvantages with the method are sucrose; high ionic strength salt

solutions and divalent metal ions cannot be entrapped efficiently [4].

B) SOLVENT DISPERSION METHODS

Figure 14: Solvent Dispersion Method.

Ethanol Injection Method

An ethanol solution of lipids is injected rapidly through a fine needle into an excess of saline or other aqueous

medium. The rate of injection is usually sufficient to achieve complete mixing. This procedure yields a high

proportion of SUVs i.e. approximately 25nm. If mixing is not done properly lipid aggregates and larger vesicles may

be formed. The major drawback of this method is limitation of the solubility of lipids in ethanol. Another drawback is

difficulty in removing residual ethanol from phospholipid membrane [4,20].

Figure 15: Preparation of Liposomes by Ethanol Injection Method [4]

Ether Injection

A solution of lipids dissolved in diethyl ether or ether/ methanol mixture is slowly injected through a narrow needle

to an aqueous solution of the material to be encapsulated at 55–65°. The subsequent removal of ether under vacuum

leads to the formation of liposomes. The main disadvantage with the method is liposomes produced are

Brought in contact with aqueous phase containing materials to be entrapped within the liposomes

Dissolved in organic solution

Lipids



heterogeneous in nature i.e. 70–190 nm and the material to be encapsulated will be exposed to higher temperature.

The efficiency of material encapsulated is relatively low. The captured volume per mole of lipid is high i.e. 8-17

litre/mol[4,20].

Figure 16: Preparation of Liposomes by Ether Injection Method [4]

3. Double Emulsion

The double emulsion is prepared by rapidly injecting the dispersion of micro droplets into hot aqueous solution of tris

buffer with the help of 22 gauge hypodermic needle under vigorous stirring. The organic solvent is evaporated using

strong jet of nitrogen thus forming double emulsion.

The traces of organic solvent are removed evaporation and finally the volume is adjusted by adding extra distilled

water and then the product is centrifuged at 20 degree Celsius for 30 minutes at 37000g to remove lipid aggregates

[4].

4. Reverse Phase Evaporation Vesicles

First water in oil emulsion is formed by sonication of a two phase system containing phospholipids in organic solvent

i.e. diethylether or isopropylether or mixture of isopropyl ether and chloroform and aqueous buffer. The organic

solvents are removed under reduced pressure, resulting in the formation of a viscous gel.

The liposomes are formed when residual solvent is removed by continued rotary evaporation under reduced pressure.

With this method high encapsulation efficiency up to 65% can be obtained in a medium of low ionic strength for

example 0.01M NaCl. The method has been used to encapsulate small and large macromolecules. The main

disadvantage of the method is the exposure of the materials to be encapsulated to organic solvents and to brief

periods of sonication [28].

5. Stable Plurilamellar Vesicles



Here, water inorganic phase dispersion is prepared with an excess of lipid followed by drying under continued bath

sonication with a discontinuous stream of nitrogen. The redistribution and equilibrium of aqueous solvent and solute

occurs in between the various bilayers in each plurilamellar vesicle. The percent entrapment is around 30% [4].

C) DETEGENT REMOVAL TECHNIQUES

Figure 17: Detergent Removal Methods

Dialysis

By lowering the concentration of detergent in the bulk aqueous phase, the molecules of detergent can be removed

from mixed micelle by dialysis.

Higher CMC indicates that the equilibrium is strongly shifted towards the bulk solution so that the removal from the

mixed membrane by dialysis becomes more easy.

Detergents commonly used here exhibit high CMC i.e. 10-20mM so that removal is facilitated. They include bile

salts: sodium cholate and sodium deoxycholate; synthetic detergents like octylglucoside[4].

Chromatography

Phospholipids in the form of either sonicated vesicles or as a dry film at a molar ratio of 2:1 with deoxycholate form

unilamellar vesicles of 100nm on removal of deoxycholate by column chromatography. This could be achieved by

passing the dispersion over a Sephadex G25 column pre saturated with constitutive lipids and pre equilibrated with

hydrating buffer [4].

3. Dilution

Mixed micellar solutions, containing constant octylglucoside and egg phosphatidylcholine concentrations and varying

amounts of cholesterol and/or a charged compound, were diluted at defined rates. After dilution, the resulting

liposome dispersions were sequentially concentrated, washed or dialyzed, and filtered.

MICELLES

Serve To Screen Hydrophobic Portions Of Molecule From Water

Detergents Associate With Phospholipids Molecule

Phospholipids + Aqueous Phase(Contact Via Detergents)



The effect of lipid composition and experimental conditions on physicochemical characteristics was studied. Fairly

homogeneous liposome dispersions with mean diameters ranging from 100 to over 200 nm could be obtained.

Liposomes with a mean diameter below 100 nm could also be obtained, but were hetero disperse and unstable [4,29].

4. Reconstituted Sendai Virus Enveloped Vesicles

Incubation of intact Sendai virions or reconstituted Sendai virus envelopes with phosphatidylcholine or cholesterol

liposomes at 37 degrees C results in virus-liposome fusion. It indicates a non leaky fusion process. Only liposomes

possessing virus receptors, namely sialoglycolipids or sialoglycoproteins, became leaky upon interaction with Sendai

virions [4,30].

II) ACTIVE LOADING

Active loading is also known as remote loading. Because limited amount of lipids can be administered systematically

and in order to deliver the drug in clinical efficacious concentrations, it is essential that liposomes are efficiently

packed with the active drug compounds.

The remote loading process is based on the transport of molecules against their concentration gradient as the

molecules are transported from bulk solution into performed liposomes.

Drug loading can be attained either passively (i.e., the drug is encapsulated during liposome formation) or actively

(i.e., after liposome formation). Hydrophobic drugs, for example amphotericin B taxol or annamycin, can be directly

combined into liposomes during vesicle formation, and the amount of uptake and retention is governed by drug-lipid

interactions. Trapping effectiveness of 100% is often achievable, but this is dependent on the solubility of the drug in

the liposome membrane. Passive encapsulation of water-soluble drugs depends on the ability of liposomes to trap

aqueous buffer containing a dissolved drug during vesicle formation [31]. Trapping effectiveness generally <30% is

limited by the trapped volume delimited in the liposomes and drug solubility. On the other hand, water-soluble drugs

that have protonizable amine functions can be actively entrapped by employing pH gradients [32], which can result in

trapping effectiveness approaching 100% [33].

Active loading methods have the following advantages over passive encapsulation techniques [4]:

A high encapsulation efficiency and capacity.

A reduced leakage of the encapsulated compounds.

CHARACTERISATION OF LIPOSOMES

I) PHYSICAL CHARACTERISTICS



VESICLE SHAPE AND LAMELLARITY

Vesicle shape can be assessed using various electron microscopic techniques. Lamellarity of vesicles i.e. the number

of bilayers present in liposomes is determined using freeze-fracture electron microscopy and 31P nuclear magnetic

resonance analysis [4,34].

Freeze Fracture and Freeze-Etch Electron Microscopy: Freeze fracture electron microscopy can be used to assess the

shape, lamellarity and surface morphology of liposomes. In this technique, the fracture plan passes through the

vesicles, which are randomly positioned in the frozen state. The observed distribution profile depends on the distance

of vesicle centre from the plane of fracture [4,34].

31p Nuclear Magnetic Resonance Analysis: This technique exploits 31P nuclear magnetic resonance NMR to

monitor the phospholipid phosphorus signal intensity. A reduction of 50% in NMR signal intensity indicates a

unilamellar vesicle whereas subsequent reduction indicates multilamellar vesicular preparation [4,34].

VESICLE SIZE AND DISTRIBUTION

Microscopic Techniques

Optical Microscopy: Vesicular dispersion are diluted and wet mounted on a haemocytometer and photographed with

a phase contrast microscope. The negatives are then projected on a piece of calibrated paper using a photographic

enlarger x1250. Diameters of approximately 500 vesicles are measured. This microscopic method includes the use of

Bright field, Phase contrast microscope and Fluorescent microscope. It is useful in evaluating the vesicle size of large

vesicles > 1 micrometer [4,8,34].

Negative Stain Transmission Electron Microscopy: Liposomes are embedded in this method in a thin film of electron

dense heavy metal stain. Negative Stain Transmission Electron Microscopy visualizes relatively electron transparent

liposomes as bright areas against a dark background [4,8,34].

Cryo Transmission Electron Microscopy Technique: This method involves freeze fracturing of the samples followed

by their visualization. Using transmission electron microscopy. Thin sample films are prepared under controlled

temperature 25 degree C and humidity conditions within a custom-built environment chamber. The films are

thereafter vitrified by quick freezing in liquid ethanol and transferred to TEM analysis [4,8,34].

Freeze Fracture Electron Microscopy

Scanning Electron Microscopy: This technique is less frequently used due to the distortion caused during the sample

preparation [4].



Diffraction And Scattering Techniques

Laser Light Scattering Techniques: It is a laser-based technique; quasi-elastic light scattering techniques are useful in

analyzing the homogenous colloids. This technique can be applied to unimodel system with diameter less than 1

micrometer [4,34].

Hydrodynamic Techniques

Field Flow Fractionation Techniques (FFF): Because of fundamental difference in driving forces, sedimentation FFF

and flow FFF measure different vesicle properties [4,34].

Gel Permeation: The ability to separate various components of the heterodispersed preparations could be exploited to

estimate various sized particles present in the dispersion. It is also used for the separation of various heterodispersed

liposomal preparations [4].

Ultracentrifuge: It yields valuable data on size distribution of liposomes. It is also used for analytical purposes [4].

3) SURFACE CHARGE

In general two methods are used to assess the charge [4],

Free flow electrophoresis

Zeta potential measurement

The surface charge can be calculated by estimating the mobility of the liposomal dispersion in a suitable buffer

determined using Helmholtz– Smolochowski equation.

4)ENCAPSULATION EFFECIENCY

It determines the percent of drug in aqueous phase is usually expressed as % entrapment/mg lipid. Encapsulation

efficiency is assessed using two techniques including mini column centrifugation method and protamine aggregation

method [4,8].

% Entrapment efficiency= Entrapped Drug (mg) X 100

Total Drug added (mg)

5) ENTRAPPED VOLUME

The entrapped volume of a population of liposomes in μl/mg phospholipids can often be deduced from measurements

of total quantity of solute entrapped inside liposome assuring that the concentration of solute in the aqueous medium

inside liposome is the same after separation from entrapped material [4].

6) PHASE RESPONSE AND TRANSITIONAL BEHAVIOR



Phase behavior of liposomal membrane determines properties such as permeability, fusion, aggregation and protein

binding. The phase transition has been evaluated using freeze fracture electron microscopy. They are more

comprehensive verified by differential scanning calorimeter analysis. Moreover these phase behaviors are important

to characterize while formulating the liposomes with the lipids having different phase transition temperatures and

polymer PEG grafted liposomes where polymer grafting as such provides a steal thing effect deterring them from

macrophagic uptake and hence long circulation [4,8].

7) DRUG RELEASE

The liposome-based formulations can be assisted by employing in vitro assays to predict pharmacokinetics and

bioavailability of the drug before employing in vivo studies, as the in vivo studies are costly and time consuming

[4,8].

II) CHEMICAL CHARACTERISTICS

PHOSPHOLIPID IDENTIFICATION AND ASSAY

Bartlett assay, Stewart assay and thin layer chromatography can be used to estimate the phospholipid concentration in

the liposomal formulation [4].

In Bartlett assay, the phospholipid phosphorus in the sample is first hydrolyzed to inorganic phosphates. This is

converted to phospho molybdic acid by addition of ammonium molybdate. Phosphor molybdic acid is quantitatively

reduced to blue colored compound by amino naphthylsulphonic acid. The intensity of blue color is measured

spectrophotometrically and is compared with the standard curve of phospholipids. The disadvantage of this method is

Bartlett assay is very sensitive to inorganic phosphates and creates problems in measurement of phospholipids.

In Stewart assay phospholipids form a complex with ammonium ferrothiocynate in organic solution. The standard

curve is first prepared by adding 0.1M ammonium ferrothiocynate solution with different concentrations of

phospholipids in chloroform. Similarly the samples are treated and optical density of the solutions is measured at

485nm and the absorbance of samples compared with standard curve of phospholipids. The advantage of this method

is that the presence of inorganic phosphates does not interfere with the assay. The disadvantage of this method is that

it is not applicable to samples where mixtures of unknown phospholipids may be present.

Thin layer chromatography is employed for determining the purity and concentration of lipids. If the compound is

pure, it should run as a single spot in all elution. Phospholipids, which are degraded, can be observed as a long smear

with a tail trailing to the origin compared to the pure material, which runs as one clearly defined spot.



2) CHOLESTROL ANALYSIS

Cholesterol oxidase assay or ferric perchlorate method and Gas liquid chromatography techniques can be used to

determine the cholesterol concentration [4].

Cholesterol is qualitatively analyzed using capillary column of flexible fused silica.

Cholesterol is quantitatively estimated by measuring the absorbance of purple complex produced with the iron upon

reaction with the combined agent containing ferric perchlorate, ethyl acetate and sulphuric acid at 610nm.

STABILITY OF LIPOSOMES

A stable dosage forms is the one which maintains the physical stability and chemical integrity of the active molecule

during its developmental procedure and storage. Hence a stability protocol is essential to study the physical and

chemical integrity of the drug product in its storage.

1. PHYSICAL STABILITY

During the storage of liposomes, the vesicles tend to aggregate and increase in size to attain thermodynamically

favorable state. Drug leakage from the vesicles can occur due to fusion and breaking of vesicles, which deteriorates

the physical stability of the liposomal drug product. Hence morphology, size and size distribution of the vesicles are

important parameters to assess the physical stability [4,10,11,16].

2. CHEMICAL STABILITY

Phospholipids are prone to oxidation and hydrolysis, which may alter the stability of the drug product. Ionic strength,

pH, solvent system and buffered species also play a major role in maintaining a liposomal formulation. Chemical

reaction can also be induced by light, oxygen, temperature and heavy metal ions. Oxidation deterioration involves the

formation of cyclic peroxides and hydroxyl peroxidases due to the result of free radical generation in the oxidation

process.

Liposomes can be prevented from oxidative degradation by protecting them from light, by adding anti-oxidants such

as alpha – tocopherol or butylated hydroxyl toluene (BHT), producing the product in an inert environment (presence

of nitrogen or Argon) or by adding EDTA to remove trace heavy metals. Hydrolysis of the ester bond at carbon

position of the glycerol moiety of phospholipids leads to the formation of lysophosphatidylcholine (lysoPC), which

enhances the permeability of the liposomal contents. Hence, it becomes necessary to control the limit of lysoPC

within the liposomal drug product. This can be achieved by formulating liposomes with phosphatidylcholine free

from lysoPC[4,10,11,16].



APPLICATIONS OF LIPOSOMES

APPLICATIONS OF LIPOSOMES IN THE SCIENCES [35]

Mathematics: Topology of two-dimensional surfaces in three-dimensional space governed only by bilayer elasticity

Physics: Aggregation behavior, fractals, soft and high-strength materials

Chemistry: Photochemistry, artificial photosynthesis, catalysis, micro compartmentalization

Biochemistry: Reconstitution of membrane proteins into artificial membranes

Biology: Model biological membranes, cell function, fusion, recognition

Pharmaceutics: Model biological membranes, cell function, fusion, recognition

Medicine: Drug-delivery and medical diagnostics, gene therapy

LIPOSOMES IN PHARMACEUTICAL INDUSTRY [35]

For solubilization amphotericin B, minoxidil are used in treatment of fungal infections.

For site-avoidance, Amphotericin B is used to reducednephro toxicity, and doxorubicin is used to decrease cardio

toxicity in fungal infections and cancer.

For sustained-release action of systemic antineoplastic drugs, hormones, corticosteroids, drug depot in the lungs in

cancer and biotherapeutics

3) LIPOSOMES AS DRUG DELIVERY VEHICLES[4]

Enhanced drug solubilization, e.g., Amphotericin B, Minoxidil, Cyclosporine

Protection of sensitive drug molecules, e.g., Cytosine arabinose, DNA, RNA

Enhanced intracellular uptake, e.g., Anticancer drugs, Antiviral drugs, Antimicrobial drugs

Altered pharmacokinetics and biodistribution i.e. prolonged or sustained release of drugs with short circulatory half-

lives.

Increased therapeutic index, e.g., Antitumor drugs i.e. cytosine arabinoside tri-phosphate (ara-CTP), Actinomycin D

4) LIPOSOMES AS A LYSOSOMOTROPIC CARRIER [4]

Liposomes have been used as lysosomotropic carriers therapeutically in enzyme diseases like Gaucher’s disease i.e.

beta glycosidase deficiency and Pompe’s disease i.e. alpha glycosidase deficiency

A variety of lysosomal enzymes can be entrapped in liposomes and administered to patients suffering from lysosomal

storage disorders.

Liposomes is also used in the treatment of metal poisoning, e.g., liposomal EDTA



5) LIPOSOMES IN ANTICANCER THERAPY[4,35]

DaunoXome®: Daunorubicin containing DSPC/chol liposomes is used in Advanced Kaposi's sarcoma

Doxil®/Caelyx®: Doxorubicin containing HSPC/chol/PEG-DSPE liposomes is used in Metastatic ovarian cancer and

advanced Kaposi's sarcoma

MyocetTM: Doxorubicin containing EPC/chol liposomes is used in Metastatic breast cancer

6) LIPOSOME AS ANTI-INFECTIVE AGENTS [20]

Active Targeting Approach

Anamycin is used in treatment of Leishmianiasis

Asiaticoside is used in treatment of Tuberculosis and Leprosy

Rifampicin is used in treatment of Tuberculosis

Passive targeting approach

Amphotericin B is used in treatment of Meningitis, Leishmianiasis, Candidiasis

Praziquantal is used in treatment of Macrophage activation

Gentamycin is used in treatment of Staphylococcal pneumonias

7) LIPOSOME IN EYE DISORDERS

Liposome has been widely used to treat disorder of both anterior and posterior segment. The disease of eye includes

dry eyes; Keratitis, an inflammation of the cornea; Corneal transplant rejection; Uveitis, An inflammation of the

middle layer of the eye; Endophthalmitis, an inflammation of the internal coats of the eye; and Proliferative

vitreoretinopathy (PVR), a disease that develops as a complication of rhegmatogenous retinal detachment i.e. retinal

separation associated with a break, a hole, or a tear in the sensory retina [20].

The liposomal drugs currently approved are ‘verteporfin’ for the use in the eye.

8) LIPOSOMES AS RADIODIAGNOSTIC CARRIERS

Liposomes are used in different imaging modalities to locate the sites specifically. Their radio diagnostic applications

include Liver and spleen imaging, Lymphatic imaging, Tumor imaging, Blood pool imaging, Brain imaging, Imaging

cardiovascular pathologies, Visualization of inflammation and infection sites, bone marrow and eye vasculature [4].

LIPOSOMES IS DERMATOLOGY AND COSMETOLOGY [4, 20]

CaptureTM

marketed by Christian Dior contains liposomes in gel with ingredients

PlenitudeTM

marketed by L’Oreal contains tanning agents in liposomes



NactosomesTM

marketed by Lancome (L’Oreal) contains vitamins, Retinol acetate, in liposomes

10) TUMOR-TARGETED ANTISENSE LIPOSOMAL THERAPY

The use of antisense oligonucleotides (asODNs) to selectively target tumor-associated genes without affecting the

normal ones appears to be a promising new approach [36].

11) FOOD APPLICATION

The ability of liposomes to solubilize compounds with demanding solubility properties, sequester compounds from

potentially harmful medium, and release incorporated molecules in a sustained and predictable way can be used in

food processing industry. A classical example is cheese making [37].

Conclusion: Since the discovery in the last six decades, liposome as a novel drug delivery systems has played a

significant role in reformulation of potent drugs to improve the therapeutics and stability. A number of drugs, which

are highly potent and have low therapeutic indication, can be targeted to the required diseased site using the

liposomal drug delivery system. Liposomal approach can be successfully utilized to improve the pharmacokinetics

and therapeutic efficacy, simultaneously reducing the toxicity of various highly potent drugs. Liposomes can cross

the blood brain barrier (BBB) because of the lipophilic nature of the phospholipids, so even the hydrophilic drugs,

which cannot easily cross the BBB, can be formulated as liposomes. Liposomes are prepared by various methods in

which the most common method applied for research purpose is film method and many more. Liposomes have a

broad range of pharmaceutical applications. Nowadays the liposomal topical formulations are mostly used in the

cosmetic and hair technologies. Liposomes are widely used as radio diagnostic agents. Liposomes are giving a good

and encouraging result in the anticancer therapy and human therapy. They are also widely used as drug delivery

vehicles, lysosomotropic carriers, anti-infective agents and in treatment of eye disorders. Liposomes are one of the

unique and most promising drug delivery system.

References

1. Mansoori and Agrawal, ‘A Review On Liposome’IJARPB, 2012; 2(4), 453-464.

2. Rutuja R D et al. ‘A Review On Liposomes’.World Journal Of Pharmacy And Pharmaceutical Sciences, 2016;

5(3), 506-517.

3. Maurer, Fenske&Cullis, ‘Developments in liposomal drug delivery systems’, Expert Opin. Biol. Ther. 2001;

1(6), 01-25.

4. Vyas SP, Khar RK Targeted and Controlled Drug Delivery: Novel Carrier Systems; 2004; 173-248.



5. Bangham A D, Horne R W. Negative Staining of Phospholipids and Their Structural Modification by Surface-

Active Agents As Observed in the Electron Microscope, Journalof Molecular Biology, 1964; 8(5), 660-668.

6. Anwekar H, Patel S, Singhai AK. Liposome as drug carriers.Int J of Pham & Life Sci 2011; 2(7); 945-51.

7. ShobhaRani,RHiremath and Vanaja K. Textbook of Industrial Pharmacy: Drug Delivery Systems, and Cosmetic

and Herbal Drug Technology 97- 120.

8. KanthShashi et al. ‘A Complete Review On Liposomes’. International Journal Of Pharmacy, 2012; 7 (3), 10-16.

9. Mazafari, M.R. Liposomes: an overview of Manufacturing Techniques, Cell Mol Bio Lett, 2005: 10: 711-719.

10. Sanjay K Jain and N K Jain, Liposomes As Drug Carriers. Controlled and Novel Drug Delivery 304-348.

11. KalepuSandeep et al, publisher. ‘Liposomal drug delivery system - A Comprehensive Review’. Int. J. Drug Dev.

& Res., 2013; 5(4) 62-75.

12. Cancer Treatment- ‘Conventional And Innovative Approaches’, Book Edited By Leticia Range 2013

13. Hiemenz PC, Rajagopalan R. Principles of colloid and surface chemistry, 3rd edition, New York: Marcel

Dekker: 1997; 120-156.

14. Kumar KS, Jyothi, Nagendar S, Devipriya S, Kumar GV, Babu S. Stability of Liposomes. Int J Adv in Pham Sci

2011; 2(4): 301-7.

15. Stryer S. Biochemistry, 1981; 213.

16. Loveleenpreetkauret al. ‘Liposome as a drug carrier – a review’.International Journal of Research in Pharmacy

and Chemistry, 2013; 3(1) 121-128.

17. Horne R W, Bangham A D, Whittaker VP.Negatively Stained Lipoprotein Membranes, Nature, 200(4913), 1963,

1340.

18. Alving CR Macrophages, as targets for delivery of liposome encapsulated antimicrobial agents, Adv Drug

Delivery Rev, 1998; 2.

19. Emanuel et al. ‘Preparation And Characterisation Of Doxorubicin Loaded Sterically Stabilized

Immunoliposomes’. Pham. Res. 1996; 13, 352-359.

20. Deepthi V and Kavitha AN. ‘Liposomal Drug Delivery System-A Review’. RGUHS J Pharm Sci. 2014; 4 (2) 47-

56.

21. Yash Roy R C. ‘Lamellar Dispersion And Phase Separation Of Chloroplast Membrane Lipids By Negative

Staining Electron Microscopy’. Journalof Biosciences, 1990; 15(2), 93-98.



22. Hope, M.Y.; Bally, M.B; Mayer, L.D. Chem. Phys. Lipids., 1986; 40, 89.

23. Samad et al.‘Liposomal Drug Delivery Systems: An Update Review’. Current Drug Delivery, 2007, 4(4), 297-

305.

24. Lasic, D.D. The mechanism of vesicle formation.Biochem J. 1988; 256: 1-11.

25. Lasic, D.D et al. ‘Spontaneous vesiculation’.Adv Colloid Interfac Sci. 2001; 89-90: 337-349.

26. Remington. The Science and Practice of Pharmacy. Vol I, 21st edition, BI Publishers Pvt Ltd; 314–6.

27. Riaz M. Review: Liposomes preparation methods. Pak J Pharm Sci. 1996; 19: 65–77.

28. J.S. Dua1, Prof. A. C. Rana, Dr. A. K. Bhandari. ‘Liposome: Methods Of Preparation And Applications’

International Journal Of Pharmaceutical Studies And Research 2012; 3(2): 14–20.

29. Jiskoot W et al ‘Preparation of liposomes via detergent removal from mixed micelles by dilution; The effect of

bilayer composition and process parameters on liposome characteristics.’ Pharm Weekbl Sci. 1986 Oct 17;8(5):

259-65.

30. V Citovsky and A Loyter. Fusion of Sendai Virions or reconstituted Sendai virus envelopes with liposomes or

erythrocyte membranes lacking virus receptors by., JBC, the journal of Biological Chemistry.

31. Akbarzadeh et al. ‘Liposome: Classification, Preparation, And Applications’. Nanoscale Res Lett.2013; 8(1):

102.

32. Mayer ID et al. 'Liposome Technology. 2. Gregoriadis G, editor. Boca Raton: CRC Press;.pH gradient-mediated

drug entrapment in liposomes; 1993; 27–44.

33. Arcadio C, Cullis PR. ‘Recent advances in liposomal drug-delivery systems’. CurrOpin Biotechnol.1995; 6, 698–

708.

34. SaraswathiMarripati. et al. ‘A Review On Liposomes’. International Journal of Research in Pharmaceutical and

Nano Sciences. 2014, 3(3), 159 - 169.

35. D.D. Lasic. Liposome Technology. ©1995 Elsevier Science B.V. Handbook of Biological Physics.Volume 1,

Edited By R. Lipowskyand E. Sackmann.Chapter 10, 491-519.

36. D Di Paolo, F Pastorino, C Brignole et al. ‘Drug Delivery Systems: Application of Liposomal Anti-Tumor

Agents to Neuroectodermal Cancer Treatment’. Tumori, 2008; 94, 246-253.

37. Laouini et al. ‘Preparation, Characterization and Applications of Liposomes: State of the Art’ Journal of Colloid

Science and Biotechnology. 2012; 1, 147–168.

https://www.ncbi.nlm.nih.gov/pubmed/3786108



Date post:	07-Feb-2018
Category:	Documents
Upload:	vukhanh
View:	219 times
Download:	1 times

ISSN: 0975 -766X CODEN: IJPTFI Available Online through ... · PDF fileNovel drug delivery...

Documents