liposomal drug delivery systems an update review

Current Drug Delivery, 2007, 4, 297-305 297

1567-2018/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

Liposomal Drug Delivery Systems: An Update Review

Abdus Samad, Y. Sultana* and M. Aqil

Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India

Abstract: The discovery of liposome or lipid vesicle emerged from self forming enclosed lipid bi-layer upon hydration; liposome drug

delivery systems have played a significant role in formulation of potent drug to improve therapeutics. Recently the liposome formulations are targeted to reduce toxicity and increase accumulation at the target site. There are several new methods of liposome preparation based

on lipid drug interaction and liposome disposition mechanism including the inhibition of rapid clearance of liposome by controlling parti-cle size, charge and surface hydration. Most clinical applications of liposomal drug delivery are targeting to tissue with or without ex-

pression of target recognition molecules on lipid membrane. The liposomes are characterized with respect to physical, chemical and bio-logical parameters. The sizing of liposome is also critical parameter which helps characterize the liposome which is usually performed by

sequential extrusion at relatively low pressure through polycarbonate membrane (PCM). This mode of drug delivery lends more safety and efficacy to administration of several classes of drugs like antiviral, antifungal, antimicrobial, vaccines, anti-tubercular drugs and gene

therapeutics. Present applications of the liposomes are in the immunology, dermatology, vaccine adjuvant, eye disorders, brain targeting, infective disease and in tumour therapy. The new developments in this field are the specific binding properties of a drug-carrying

liposome to a target cell such as a tumor cell and specific molecules in the body (antibodies, proteins, peptides etc.); stealth liposomes which are especially being used as carriers for hydrophilic (water soluble) anticancer drugs like doxorubicin, mitoxantrone; and bisphos-

phonate-liposome mediated depletion of macrophages. This review would be a help to the researchers working in the area of liposomal drug delivery.

Keywords: Liposome, characterization, sizing of liposomes, surface hydration, targeted site, phospholipids.

INTRODUCTION

When phospholipids are dispersed in water, they spontaneously form closed structure with internal aqueous environment bounded by phospholipid bilayer membranes, this vesicular system is called as liposome [1]. Liposomes are the small vesicle of spherical shape that can be produced from cholesterols, non toxic surfactants, sphingolipids, glycolipids, long chain fatty acids and even mem-brane proteins [2]. Liposomes are the drug carrier loaded with great variety of molecules such as small drug molecules, proteins, nu-cleotides and even plasmids. Liposomes were discovered about 40 years ago by A.D. Bangham [3] which has become the versatile tool in biology, biochemistry and medicine today. In 1960s, liposome has been used as a carrier to deliver a wide variety of compounds in its aqueous compartment. Liposome can be formulated and proc-essed to differ in size, composition, charge and lamellarity. To date liposomal formulations of anti-tumor drugs and antifungal agents have been commercialized [4].

The clinical potential of liposomes as a vehicle for replacement therapy in genetic deficiencies of lysosomal enzymes was first demonstrated in 1970s [5, 6].

Considerable progress was made during 1970s and 1980s in the field of liposome stability leading to long circulation times of liposomes after intravenous administration resulting in the im-provement in bio-distribution of liposome.

The important anti-tumour drug doxorubicin had been formu-lated as liposome in 1980s to improve the therapeutic index.

There are several mechanisms by which liposomes act within and outside the body which are as follows [7]:

1- Liposome attaches to cellular membrane and appears to fuse with them, releasing their content into the cell.

2- Some times they are taken up by the cell and their phosphol-ipids are incorporated into the cell membrane by which the drug trapped inside is released.

*Address correspondence to this author at the Department of Pharmaceutics,

Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India; Tel: +919899078661; +919811539489; E-mail: [email protected],

[email protected]

3- In the case of phagocyte cell, the liposomes are taken up, the phospholipid walls are acted upon by organelles called lysosomesand the active pharmaceutical ingredients are released.

Classification of Liposomes [8]

Liposomes are classified on the basis of:

1. Structure.

2. Method of preparation.

3. Composition and application.

4. Conventional liposome.

5. Specialty liposome.

1. Classification Based on Structure (Table 1)

Table-1. Vesicle Types with their Size and Number of Lipid Layers

Vesicle Type Abbreviation Diameter Size No 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 100nm One

Giant Unilamellar

vesicle

GUV More than 1 micro

meter

One

Oligolamellar vesicle OLV 0.1-1 micro meter Approx. 5

Multilamellar vesicle MLV More than 0.5 5-25

Multi vesicular vesicle MV More than 1 micro

meter

Multi com-

partmental

structure

Not For Distribution

298 Current Drug Delivery, 2007, Vol. 4, No. 4 Samad et al.

2. Based on Method of Preparation (Table 2)

Table-2. Different Preparation Methods and the Vesicles Formed by

these Methods

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 pluri lamellar vesicle SPLV

Frozen and thawed multi lamellar vesicle FATMLV

Vesicle prepared by extrusion technique VET

Dehydration- Rehydration method DR V

3. Based on Composition and Application (Table 3)

Table-3. Different Liposome with their Compositions

Type of Liposome Abbreviation Composition

Conventional liposome CL Neutral or negatively charge phos-

pholipids and cholesterol

Fusogenic liposome RSVE Reconstituted sendai virus envelops

PH sensitive liposomes - Phospholipids such as PER or DOPE

with either CHEMS or OA

Cationic liposome - Cationic lipid with DOPE

Long circulatory

liposome

LCL Neutral high temp, cholesterol, and 5-

10% PEG, DSP

Immuno liposome IL CL or LCL with attached monoclonal

antibody or recognition sequences

Based Upon Conventional Liposome

1- Stabilize natural lecithin (PC) mixtures

2- Synthetic identical, chain phospholipids

3- Glycolipids containing liposome

Based Upon Speciality Liposome

1- Bipolar fatty acid

2- Antibody directed liposome.

3- Methyl/ Methylene x- linked liposome.

4- Lipoprotein coated liposome.

5- Carbohydrate coated liposome.

6- Multiple encapsulated liposome.

PREPARATION OF LIPOSOMES

An important parameter to be considered when preparing the liposome is the rigidity of bi-layers. There are several groups of phospholipids that can be used for the liposome preparation which are as follows:

1. Phospholipids from natural source

2. Phospholipids modified from natural source

3. Semi synthetic phospholipids

4. Fully synthetic phospholipids and

5. Phospholipids with natural head groups

Dilauryl phosphotidyl choline (DLPC), Dimyristoyl phosphoti-dyl choline (DMPC), Dipalmitoyl phosphotidyl choline (DPPC), Distearoyl phosphotidyl choline (DSPC), Dioleolyl phosphotidyl choline (DOPC), Dilauryl phosphotidyl ethanolamine (DLPE), Dimyristoyl phosphotidyl ethanolamine (DMPE), Distearoyl phos-

photidyl ethanolamine (DSPE), Dioleoyl phosphotidyl ethanola-mine (DOPE), Dilauryl phosphotidyl glycerol (DLPG), Distearoyl phosphotidyl serine (DSPS) are the commonly used phospholipids for liposome preparation (Table 4). Cholesterol can be added to the bilayers mixture for the following purposes:

1. Act as a fluidity buffer

2. Act as intercalator with phospholipids molecules

Alters the freedom of formation of carbon molecule in the acyl chain

3. Restrict the transformation of Trans to gauche conformation.

Rajendran et al. [9] prepared liposome from egg lecithin by modified ether injection technique using stearylamine added dice-tylphosphate as the charge inducing agent. The charged liposomes were larger in the size and showed better drug entrapment effi-ciency and were found more entrapped in the organs of the reticulo-endothelial system than neutral liposome.

It was also shown that cholesterol decreased the permeability coefficients of negative, neutral as well as positively charged mem-branes to Na

+, K

+, Cl

- and glucose. Cholesterol also stabilized the

membranes against temperature changes, leading to lower perme-ability at elevated temperatures [11].

Cholesterol is essential for lowering membrane permeability, and imparting better stability. Cholesterol also modulates mem-brane-protein interactions. The polymorphic phase behavior of lipids is modulated by a variety of factors including hydration, tem-perature and divalent cations, degree of unsaturation of the acyl chains and the presence of other lipids such as sterols [12, 13].

Lasic et al. [14] tried active trapping of amphipathic weak bases inside liposomes by using pH induced transmembrane potential establishment. Doxorubicin was used as a model drug.

Triton X-100 has been observed to affect the physical proper-ties of liposomes [15]. Solubilizing effect of Triton X-100 on the membranes composed of different phospholipids and cholesterol has been evaluated [16]. The measured changes indicated a drastic structure transformation into entities of fairly high dipolar moment leading to solubilization of phospholipids membranes.

The three different strategies for the preparation of liposomes are as follows:

1. Mechanical methods

2. Methods based on replacement of organic solvent

3. Methods based on size transformation or fusion of prepared vesi-cle

1. MECHANICAL METHODS

A. Film Method

The original method of Bangham et al. [2] is still the simplest procedure for the liposome formation but having some limitation because of its low encapsulation efficiency. In this technique liposome are prepared by hydrating the thin lipid film in an organic solvent and organic solvent is then removed by film deposition under vacuum. When all the solvent get removed, the solid lipid mixture is hydrated using aqueous buffer. The lipids spontaneously swell and hydrate to form liposome. This methods yields a hetero-geneous sized population of MLVs over 1 micro meter in diameter.

Nagarsenkar et al. [17] prepared lidocaine encapsulated liposomes employing the conventional lipid film hydration tech-nique. The prepared liposomal dispersions were investigated, the results showed that lidocaine incorporated into the liposomes got selectively partitioned and localized in the skin to a greater extent. A topical liposomal gel formulation containing 2% w/w lidocaine was prepared using cabopol-934 as the gelling agent. The liposome preparation of lidocaine gave a much longer duration of action compared to the conventional topical formulation.


Liposomal Drug Delivery Systems Current Drug Delivery, 2007, Vol. 4, No. 4 299

Ridy et al. [18] prepared oxamniquine liposomes with different compositions and surface charge were prepared by the chloroform film method. The amount of oxamniquine entrapped was estimated. Negatively charged liposomes exhibited the highest percentage entrapment (23.09%). The maximum oxamniquine entrapment was achieved in liposomes prepared from phospholipid molar ratio (7:4:1). The chemoprophylactic effect of free and oxamiquine liposome formulations was estimated and showed that drug encap-sulated in liposomes provided more efficient prophylaxis.

B. Ultrasonic Method

This method is used for the preparation of SUVs with diameter in the range of 15-25 m. Ultrasonication of an aqueous dispersion of phospholipids is done by two types of sonicators i.e. either probe sonicators or bath sonicators. The probe sonicators are used for the small volume which requires high energy while the bath sonicators are employed for the large volume [19].

2. METHODS BASED ON REPLACEMENT OF ORGANIC SOLVENTS

In this method lipids are co-solvated in organic solution, which is then dispersed into aqueous phase containing material to be en-trapped within the liposome. This method is of two types:

A. Reverse Phase Evaporation

The lipid mixture is added to a round bottom flask and the sol-vent is removed under reduced pressure by a rotary evaporator. The system is purged with nitrogen and lipids are re-dissolved in the organic phase which is the phase in which the reverse phase vesicle will form. Diethyl ether and isopropyl ether are the usual solvents of choice. After the lipids are redissolved the emulsion are obtained and than the solvent is removed from an emulsion by evaporation to a semisolid gel under reduced pressure. Non encapsulated material is then removed. The resulting liposomes are called reverse phase evaporation vesicles (REV). This method is used for the preparation

of large uni-lamellar and oligo-lamellar vesicles formulation and it has the ability to encapsulate large macromolecules with high effi-ciency [20].

Pleumchitt et al. [21] showed the physicochemical properties of phospatidylcholine cholesterol liposomes containing amphotericin B prepared by reverse phase evaporation method. The liposomes containing amphotericin B 2.0 mol % of total lipid demonstrated the highest percentage of drug entrapment. The highest drug en-trapment efficiency (approx. 95%) with particle size range of 1307-1451 nm was obtained with the formulation containing 1:1 molar ratio of phosphatidycholine to cholesterol.

B. Ether Vaporization Method

There are two method according to the solvent used:

i. Ethanol injection method.

ii. Ether injection method.

In ethanol injection method, the lipid is injected rapidly through a fine needle into an excess of saline or other aqueous medium. In ether injection method the lipid is injected very slowly through a fine needle into an excess of saline or other aqueous medium.

3. METHODS BASED ON SIZE TRANSFORMATION OR FUSION OF PREFORMED VESICLE

A. Freeze Thaw Extrusion Method

The freeze thaw method is an extension of the classical DRV method. Liposomes formed by the film method are vortexed with the solute to be entrapped until the entire film is suspended and the resulted MLVs are frozen in luke warm water and than vortexed again. After two cycles of freeze thaw and vortexing the sample is extruded three times. This is followed by six freeze thaw cycle and addition eight extrusions. This process ruptures and defuses SUVs during which the solute equilibrates between inside and outside and liposome themselves fuse and increase in size to form large Uni lamellar vesicle by extrusion technique (LUVET). For the encapsu-lation of protein this method is widely used [22].

Table 4. Phospholipids and Cholesterol which are Used for the Preparation of Liposome and their Transition Temperature (Tc) and Molecular

Weights [10]

Phospholipids Abbreviation Charge Tc (0c) Mol. Wt.

Brain Sphingomyellin BSPM 0 32 814

Cholesterol CH 0 -- 387

Dimyristoyl phosphatidylcholine DMPC 0 23 678

Dipalmitoyl phosphatidylcholine DPPC 041 734

Distearoyl phosphatidylcholine DSPC 0 58 790

Dioleyl phosphatidylcholine DOPC 0 -22 786

Dilaruryl phosphatidylglycerol DLPG -1 4 633

Dimyristoyl phosphatidylglycerol DMPG -1 23 689

Dipalmitoyl phosphatidylglycerol DPPG -1 41 745

Distearoyl phosphatidylglycerol DSPG -1 55 796

Phosphatidyl ethanolamine PE 0 48 720

Dimyristoyl phosphatidyl ethanolamine DMPE 0 -- 636

Dipalmitoyl phosphatidyl ethanolamine DPPE 0 60 692

Distearoyl phosphatidyl ethanolamine DSPE 0 -- 748

Dimyristoyl PA DMPA -2 51 618

Dipalmitoyl PA DPPA -2 67 649

Dioleoyl PA DOPA -2 -- 701

Dipalmitoyl phosphatidyl serine DPPS -- 48 758

Dicetyl phosphate DCP -1 -- 547



Canto et al. [23] prepared liposomes of soya phosphatidylcho-line, cholesterol and stearylamine (molar ratio 6/3/1) and 0.1 % to-copherol by the extrusion of multi-lamellar vesicles through 0.2

m polycarbonate membrane. They analyzed free piroxicam at 4 mg/kg, piroxicam encapsulated in liposomes and added to 1.5% hydroxyethylcellulose (HEC) gel at 1.6 mg/kg, and piroxicam en-capsulated in liposomes and added to HEC gel at 4 mg/kg. The inhibition of inflammation obtained was 21.1%, 32.8% and 47.4% respectively. These results showed that the encapsulation of piroxi-cam produced an increase of topical anti-inflammatory effect.

B. The Dehydration- Rehydration Method

In this method the empty buffer containing SUVs and rehydrat-ing it with the aqueous fluid containing the material to be entrapped after which they are dried. This leads to a dispersion of solid lipids in finely subdivided form. Freeze drying is often the method of choice. The vesicles are than rehydrated. Liposomes obtained by this method are usually oligo lamellar vesicle [24].

Bhalerao et al. [25] prepared liposome by the conventional thin film hydration technique. The result showed that the formation of bi-layered liposomes in the particle size range of 0.2-0.8276 moccurred with a maximum entrapment efficiency of 42.6%. The liposomes stored at 4-5

oC showed maximum stability as compared

to those stored at any other temperature.

SIZING OF LIPOSOMES:

Size characteristic of liposome have a major effect on their fate i.e. for which application they can be used for. The therapeutic applications of liposome are dependent on physical integrity and stability of lipid bilayers structure. Therefore liposome production procedure must be predictable and reproducible with particle size distribution within a certain size range. Lipid based formulation can be devised as site specific drug delivery vesicles that are:

1. Relatively cleared by kupffer cells of liver and the macro-phages [26].

2. Evade detection of active substance by the reticulo endothe-lial system (RES) and efficiency deliver liposome incorporated material to target tissue, organ or tumor [27]. Sizing of liposome are usually performed by sequential extrusion at relatively low pressure through polycarbonate membrane (PCM). The membrane extrusion technique can be used to process LUVs as well as MLVs to form LUVETs. The membrane (PCM) is of pore size 0.27 micrometer. The other procedure of sizing of the liposomes are gel chromatog-raphy , mainly used to size liposome but more typically used to remove encapsulated components by separation. Sonication is the third method of sizing of liposomes, but this method is related with following disadvantages:

1. Exclusion of oxygen is difficult which result in per oxidation reaction

2. Titanium probes shed metal particle resulting in contamination

3. They can generate aerosols, which exclude them with from use with certain agents

These above problems are mainly related with the probe sonica-tion but these problems can be removed by using the bath sonica-tion.

CHARACTERIZATION OF LIPOSOMES (TABLE 5)

After preparation and before use in immunoassay the liposome must be characterized. Evaluation could be classified into three broad categories which are physical, chemical and biological meth-ods. The physical methods include various parameters, which are size, shape, surface features, lamellarity phase behaviors and drug release profile.

Ma et al. [28] evaluated structural integrity of liposomal phos-pholipids membrane by a new technique of gamma-ray perturb

angular correlation (PAC) spectroscopy. In this 111

In-label di-ethyene triamine penta acetic acid (DTPA) derivative dipalmitoyl phosphatidyl ethanolamine (DPPE) lipid were incorporated in the SUVs. This helped in the continuous non- invasive monitoring of the microenvironment of the lipid bilayer.

Kolchens [29] used quasi elastic light scattering (QELS) or photon correlate spectroscopic (PCS) technique for determining distribution of vesicles prepared by freeze-thaw extrusion method. The influence of filter pore size, extrusion press and lipid concen-tration on the size and size distribution extruded vesicles was stud-ied.

Chemical characterization includes those studies which estab-lished the purity and potency of various liposomal constituents.

Biological characterization is helpful in establishing the safety and suitability of formulation for the in vivo use for therapeutic application [30]. The characteristics of the carrier through appropri-ate choice of membrane components, size and charge determines the final behavior of liposomes both in vitro and in vivo as well [31, 32].

Jousma et al. [33] characterized the vesicles obtained by repeti-tive extrusion through polycarbonate membranes using freeze-fracture electron microscopy, small angle X-ray scattering (SAXS), and encapsulated volume using 6-carboxy flurescein (6-CF) and 31

p-NMR as aqueous markers.

Table-5. Characterization of Liposomes with their Quality Control

Assays [34-52]

A. Biological characterization

Characterization parameters Instrument for analysis

Sterility Aerobic/anaerobic culture

Pyrogenicity Rabbit fever response

Animal toxicity Monitoring survival rats

B. Chemical characterization

Characterization parameter Instrument for analysis

Phospholipids concentration HPLC/Barrlet assay

Cholesterol concentration HPLC / cholesterol oxide assay

Drug concentration Assay method

Phospholipids per oxidation UV observance

Phospholipids hydrolysis HPLC/ TLC

Cholesterol auto-oxidation HPLC/ TLC

Anti-oxidant degradation HPLC/TLC

PH PH meter

Osmolarity Osmometer

C. Physical Characterization

Characterization parameter Instrument for analysis

Vesicle shape, and surface morphology TEM and SEM

Vesicle size and size distribution Dynamic light scattering ,TEM

Surface charge Free flow electrophoresis

Electrical surface potential and surface

PH

Zeta potential measurement and PH

sensitive probes

Lamellarity P31 NMR

Phase behavior DSC, freeze fracture electron micros-

copy

Percent capture Mini column centrifugation, gel exclu-

sion

Drug release Diffuse cell/ dialysis



STABILIZATION OF LIPOSOME

The stability of liposome should meet the same standard as conventional pharmaceutical formulation. The stability of any pharmaceutical product is the capabilities of the delivery system in the prescribed formulation to remain within defined or pre estab-lished limits for predetermined period of time. Chemical stability involves prevention of both the hydrolysis of ester bonds in the phospholipids bilayers and the oxidation of unsaturated sites in the lipid chain. Chemical instability leads to physical instability or leakage of encapsulated drug from the bilayers and fusion and fi-nally aggregation of vesicles [53].

Chen et al. introduced the pro-liposome concept of liposome preparation to avoid physicochemical instability encountered in liposome suspension such as aggregation, fusion, hydrolysis, and/or oxidation [54].

Approaches that can be taken to increase liposomal stability involve efficient formulation and lyophillization. Formulation in-volves the selection of the appropriate lipid composition, concentra-tion of bilayers, aqueous phase ingredients such as buffers, antioxi-dant, metal chelators and cryo protectants. Charge inducing lipid such as phosphotidyl glycerol can be incorporated into liposome bilayers to decrease fusion while cholesterol and sphingomyellin can be included in the formulation to decrease permeability and leakage of encapsulated drugs. Buffers at neutral pH can decrease hydrolysis; addition of antioxidant such as sodium ascorbate can decrease oxidation. Oxygen potential is kept to minimum during processing by nitrogen purging solution [55].

In general successful formulation of stable liposomal drug product requires the following precautions:

1. Processing with fresh, purified lipids and solvents.

2. Avoidance of high temperature and excessive shear forces

3. Maintenance of low oxygen potential (Nitrogen purging)

4. Use of antioxidant or metal chelators

5. Formulating at neutral pH.

6. Use of lyo-protectant when freeze drying

APPLICATION OF LIPOSOME

The field of liposome research has expanded considerably over last 30 years. It is now possible to engineer a wide range of liposome of varying size, phospholipids composition, cholesterol composition, surface morphology suitable for wide range of appli-cation [56].

Liposomes interact with cells in many ways to cause liposomal components to be associated with target cells [7].

The liposome carrier can be targeted to liver and spleen and distinction can be made between normal and tumors tissue using tomography. In case of transdermal drug delivery system, liposome has a great application. Liposomal drug delivery system when used to target the tumor cells leads to reduction in the toxic effect and enhances the effectiveness of drugs. The targeting of the liposome to the site of action takes place by the attachment of amino acid fragment, such as antibody or protein or appropriate fragments that target specific receptors cell. Liposomal DNA delivery vectors and further enhancement in the form of LPDI -I and LPD-II are some of the safest and potential most versatile transfer vectors which are used to date.

DNA vaccination and improved efficiency of gene therapy are just a few of the recent application of liposome. Several modes of drug delivery application have been purposed for the liposomal drug delivery system, few of them are as follows:

1. Enhance drug solublisation (Amphotericin-B, Minoxidil, Pacli-taxels, and Cyclosporins)

2. Protection of sensitive drug molecules (Cytosine arabinosa, DNA, RNA, Anti-sense oligo-nucleotides, Ribozymes)

3. Enhance intracellular uptake (Anticancer, anti viral and antimi-crobial drugs)

4. Altered pharmacokinetics and bio-distribution (prolonged or sustained released drugs with short circulatory half life)

Several recent applications of liposomal drug delivery system are as follows:

A. Liposome for Respiratory Drug Delivery System

Liposome is widely used in several types of respiratory disor-ders. Liposomal aerosol has several advantages over ordinary aero-sol which are [57] as follows:

1. Sustained release

2. Prevention of local irritation

3. Reduced toxicity and

4. Improved stability in the large aqueous core.

Several injectable liposome based product are now in the mar-ket including ambisome, Fungisome and Myocet. To be effective, liposomal drug delivery system for the lung is dependent on the following parameters:

1. Lipid composition

2. Size

3. Charge

4. Drug and Lipid ratio and

5. Method of delivery

The recent use of liposome for the delivery of DNA to the lung means that a greater understanding of their use in macromolecular delivery via inhalational is now emerging. Much of this new knowledge, including new lipids and analytical techniques, can be used in the development of liposome based protein formulations. For inhalation of liposome the liquid or dry form is taken and the drug release occurs during nebulization. Drug powder liposome has been produced by milling or by spry drying. Drugs which are for-mulated in the form of liposome are presented in Table 6.

Table-6. Liposomal Formulation for the Respiratory Disorder [58]

Active constituent Effect

Insulin Facilitated pulmonary adsorption and enhanced hypo-

glycemic effect

Catalase Conferred resistance to pulmonary oxygen toxicity

Super oxide dusmutase Minimize toxicity to subsequent hyperoxia and im-

proved survival

Cyclosporins Preferentially adsorbed by lung and shows sustained

release

Ricin vaccine Improved safety profile for intra pulmonary vaccina-

tion

Interleukin-2 The lungs Facilitated bioactivity and reduce toxicity

Isoniazid and Rifam-

picin

Improved the effect of drugs for the tuberculosis

B. Liposome in Nucleic Acid Therapy

Recombinant DNA technologies and studies of gene function and gene therapy all depends on the successful delivery of nucleic acid into cells in vitro and in vivo. Non viral vectors will be devel-oped for the selective delivery of the gene to the malignant cells. The vector will exploit the increase requirements of rapidly grow-ing cells for more nutrients by attaching a nutrients ligand onto the vector (liposome). The vector additionally will have a passively charged lipid to enhance nucleic acid binding along with novel pH



sensitive surfactants. The advantages and disadvantages of these technologies are given in Table 7. The role of surfactants is to in-crease the amount of nucleic acid escaping the endosome and corre-spondingly increase the transfection efficiency [59, 60]. The vector system will be evaluated first in an animal model of cancer. Lipid based gene delivery is the focus of several specialized high tech-nology companies, of which Vical ( San Diago, CA, USA), Gen-zyme ( Farmington, MA, USA) and Megabios (Burhingam, CA, USA) have products in clinical trials.

Preliminary studies will be carried out using a marker gene (BETA GALCTOSIDASE) with later experiments using a gene encoding for cytosine deaminase. Cytosine deaminase can catalyze the conversion of the innocuous agent 5- Flourocytosine to the anti-cancer agent 5- Fluorouracil. By selective delivery of this gene to only cancer cells, the therapeutic index of 5-Flourouracil can then be increased. Some of the engineered liposomal and non liposomal versions like pH sensitive cationic and anionic liposome, pH sensi-tive immuno-liposome, fusagenic liposome, genosomes, lipofec-tions and recently cochleats are being investigated as the major gene vectors (Table 8). This agent will become activated at en-dosomal pH, has membrane disrupting effects and will be inacti-vated before reaching the lysosome [61]. For this agent to work, the surfactant must enter the cell by endocytosis. The soft surfactant will be incorporated into liposome that has shown to be entering cell in this manner. The novelty of this delivery system stems from soft pH sensitive surfactants (SPS). The pH sensitive liposomes have been reported as plasmid expression vectors for the cytosolic delivery of DNA. It is also effective carrier for intracellular traffick-ing of anti sense oligo nucleotides [62].

Table-7. Advantages and Disadvantages of Recombinant Technologies

Types of Vectors Advantage Disadvantage

Viral vectors ( Adeno

virus, Retro vrus and

Adeno-associated virus)

Relative high transfec-

tion efficiency

Immunogenicity, pres-

ence of contaminants

and safety

Vector restricted size

limitation for recombi-

nant gene

Unfavourable pharma-

ceutical issue

Non viral vectors

(Liposome, polymers

and peptides)

Favourable pharmaceu-

tical issue

Plasmid ind4ependent

structure

Low immunogenicity

Opportunity for chemi-

cal / physical manipula-

tion

Relatively low transfec-

tion efficiency

Table-8. Some of the Widely Cationic Liposome Formulations

Name Composition

Lipofectin DOTMA:DOPE(1:1)

Lipofetamine DOSPA:DOPE(3:1)

LipofectASE DDAB:DOPE(1:2.1)

TranfectASE DDAB:DOPE(1:3)

Transfectam DOGS

DC-chol DC-Chol:DOPE(3:2)

DOTMA:N-[1-(2,3-Dioleoyloxy)propyl]N,N,N-trimethyl ammonium chloride

DOPE: Dioleoylphosphatidylethanolamine

DOSPA:2,3-Dioleoyloxy[2(sperminecarboxamido)ethyl]N,N- dimethyl-1-propanammonium triflou-

roacetate

DDAB: Dimethyl-dioctadecylethanolamine

DOGS: Dioctadecylamido glycyl spermine

DC-Chol: Chol-dimethyl amino ethane carbamoyl cholesterol.

C. Liposome in Eye Disorders

Liposome has been widely used to treat disorder of both ante-rior and posterior segment. The disease of eye includes dry eyes, keratitis, corneal transplant rejection, uveitis, endopthelmitis and proliferative vitro retinopathy. Retinal diseases are leading cause of blindness in advanced countries. Liposome is used as vector for genetic transfection and monoclonal antibody directed vehicle. The recent techniques of the treatment like applying of focal laser to heat induced release of liposomal drugs and dyes are used in the treatment of selective tumour and neo-vascular vessels occlusion, angiography, retinal and choroidal blood vessel stasis. Liposomal drug formulations have been approved for the two of patent drugs to date and several other products are under clinical trials. The liposomal drugs currently approved are ‘verteporfin’ for the use in the eye , the benefit of the liposome will be applied in treatment, diagnostic and research aspect of ophthalmology in future [63].

D. Liposome as Vaccine Adjuvant [64]

Liposome has been firmly established as immuno-adjuvant, potentiating both cell mediated and non cell mediated (humoral) immunity (Table 9).

Liposome acts as immuno-adjuvant by the following therapeu-tic points of view:

1. Liposomes as an immunological (vaccine) adjuvant

2. Liposomal vaccines

3. Liposomes as carrier of immuno modulation for

4. Liposomes as a tool in immuno diagnostics

Liposomal immuno-adjuvant act by slowly releasing encapsu-lated antigen on intramuscular injection and also by passively ac-cumulating within regional lymph node [65].

The accumulation of liposome to lymphoid is done by the tar-geting of liposome with the help of phosphotidyl serine. Liposomal vaccine can be prepared by inoculating microbes, soluble antigen, cytokinesis of deoxyribonucleic acid with liposome. The latter stimulating an immune response on expression of antigenic protein. Antigens can be covalently coupled to liposomal membrane [66].

Table-9. Some Antigens as Liposomal Preparation and their Appli-

cations

Antigens as Liposomal Preparation Applications

Rabies glycoproteins Interleukin -2 enhancement

Cholera toxin Enhanced Ab* level

Diphtheria toxoid Superior immunoadjuvant

Herpes simplex virus Enhanced Ab level

Hepatitis B virus Higher Ab response

Bacterial polysaccharides Superior immunoadjuvants

Tetanus toxoids Increased Ab titre

Influenza subunit antigen Intranasal, protects animal from virus

Carbohydrate antigen Increased Ab titre in salivary gland

*Ab = Antibody

Liposomes which are encapsulating antigen are second time encapsulated within alginate lysine microcapsules, to control anti-gen release and improve the antibodies responses. Liposomal vac-cines can be store at the refrigerated condition for about 12 months.

E. Liposomes for Brain Targeting

The biocompatible and biodegradable behaviour of liposomes have recently led to their exploration as drug delivery system to brain [67].



Liposomes with a small diameter (100 nm) as well as large diameter undergo free diffusion through the Blood Brain Barrier (BBB). However it is possible that a small unilamellar vesicles (SUVS) coupled to brain drug transport vectors may be transported through the BBB by receptor mediated or absorptive mediated tran-scytosis. Similarly, cationic liposomes which were developed re-cently showed these structures to undergo absorptive mediated endocytosis into cells. Whether cationic liposomes successfully undergo absorptive mediated transcytosis through the BBB has not yet been determined. The transport of substances through BBB by liposomes was extensively studied. The important finding issues from their studies are that the addition of the sulphatide (a sulphur ester of galactocerebroside) to liposome composition increases their several recent applications ability to cross BBB [68]. Wang et al.reported that liposomes coated with the mannose reach brain tissue and the mannose coat assist transport of loaded drug through BBB [69]. The neutropeptides, leu-enkephaline and mefenkephalin kyo-forphin normally do not cross BBB when given systemically. The anti depressant amitriptylline normally penetrate the BBB, due to versatility of this method. Nanoparticles (NP) were fabricated with different stabilizers. It was found that amitriptyline level was sig-nificantly enhanced in brain when the substance was adsorbed onto the NP and coated or particle stabilized with polysorbate 85 [70].

F. Liposome as Anti-Infective Agents

Intracellular pathogen like protozoal, bacterial, and fungal re-side in the liver and spleen and thus to remove these pathogen the therapeutic agent may be targeted to these organ using liposome as vehicle system [71]. The disease like leishmaniasis, candidiasis, aspergelosis, histoplasmosis, erythrococosis, gerardiasis, malaria and tuberculosis are targeted by the respective therapeutic agent using liposome as carrier shown in Table 10.

Table-10. Some Liposomal Preparation for Infective Disease

Active Constituents Application

Active targeting approach

Pentamidin Leishmaniasis

Antisense oligo-nucleotides Leishmaniasis

Anamycin Leishmaniasis

Asiaticoside Tuberculosis and Leprosy

Rifampicin Tuberculosis

Passive targeting approach

Amphotericin B Meningitis, Leishmaniasis, Candidiasis

Praziquantal Macrophage activation

Sparfloxacin M. avium, M. Intracellularie complex

Gentamycin Staphylococcal pneumonias

Use of Amphotericin B, a polyene antibiotic, in the treatment of systemic fungal infection is associated with extensive renal toxicity. Amphotericin B act by the mechanism, in which it binds to sterol in the membrane of sensitive fungi, thus increasing the membrane permeability. The toxicity of this compound is due to non specific-ity and binding to the mammalian cell cholesterol. Recently the first preparation of Amphotericin B (ambisome) in the form of liposome had passed all clinical trials and now it is used for the treatment of fungal infections. Liposomal Amphotericin B, by passively target-ing the liver and spleen, reduces the renal and general toxicity at normal dose but renal toxicity appears when the drug is given at elevated dose due to the saturation of liver and spleen macrophages. Liposome can also be targeted to lungs by coating vesicle with o-stearoyl amylopectin, polyoxylethylene or mono-sialogangliocyte. The encapsulation of antitubercular agent like isoniazid and rifam-

picin in lung targeted liposome modulates the toxicity and improve the efficiency of these drugs [72, 73]. Various formulations of the Amphotericin (Table 11) had been approved by several clinical trials and are now marketed at different European countries [74].

Table-11. Various Liposomal Preparation of Amphotericin B

Preparation Drug Targeted Site

Liposome (Am Bisome) Amphotericin B Systemic fungal infection,

Visceral lieshmaniaisis

Liposome (Amphocil) Amphotericin B Systemic fungal infection

Liposome (ABLC) Amphotericin B Systemic fungal infection

LIPOSOME IN TUMOUR THERAPY

The long term therapy of anticancer drug leads to several toxic side effect. The liposomal therapy for the targeting to the tumour cell have been revolutionized the world of cancer therapy with least side effect. It has been said that the small and stable liposome are passively targeted to different tumour because they can circulate for longer time and they can extra vasate in tissue with enhanced vas-cular permeability [75, 76]. Liposome macrophage uptake by liver and spleen hampered the development of liposome as drug delivery for aver 20 years.

Several formulations of liposomal anticancer drug which are in clinical use are given in Table 12.

Table-12. Various Intravenous Liposomal Antibiotics / Anti-

Neoplastics

Preparation Drug Targeted Site

Liposome (Doxil) Doxorubicin Kaposi’ sarcoma

Liposome

(EVACTTM)

Doxorubicin Refractory tumour

Metastatic breast cancer

Liposome

(DaunoXome)

Daunosome Advanced Kaposi’ sarcoma, breast ,

small cell lung cancer, leukaemia and

solid tumour

Liposome Nystatin Systemic fungal infection

Liposome Anamycin Kaposi’ sarcoma, Refractory breast

cancer

Liposome (Vin-

caXome)

Vincristine Solid tumour

Liposome ( Mika-

some)

Amikacin Serious bacterial infection

Doxil is the liposomal formulation of doxorubicin, intravenous, chemotherapeutic agent. Doxil is prepared by the new technology called stealth technology, stealth liposome. These are the long cir-culatory liposome which is prepared by several means. Caelyx and myocet are the liposomal formulations of doxorubicin. Caelyx is used for treatment of metastatic ovarian cancer but now in now in advanced breast cancer. Myocet s approved for metastatic breast cancer [77-79].

CONCLUSION

Almost from the time of their discovery in 1960’s and the dem-onstration of their entrapment potential, liposome vesicle have drawn attention of researchers as potential carriers of various bioac-tive molecules that could be used for therapeutic applications in human and animals. Many factors contribute to their success as drug delivery vehicles. Liposomes solubilise lipophillic drug candi-dates that would otherwise be difficult to administer intravenously. The encapsulated drug is inaccessible to metabolizing enzyme; conversely, body component such as erythrocyte and tissue injec-tion site are not directly exposed to full dose of the drug. Liposomes



can cross the BBB because of the lipophillic nature of the phosphol-ipids, so even the hydrophilic drugs (which otherwise can not easily cross the BBB) might be formulated as liposomes. Liposome can prolong the drug action by slowly releasing the drug in the body. Targeting option change the distribution of the drug in the body. They can also be used as adjuvant in vaccine formulation.

Liposome are prepared by various methods in which the most common method applied for research purpose are film method and dehydration rehydration method and many more. Stabilization of liposomes has been an area of concern for optimum shelf life of the liposomal formulation. Nowadays, stealth liposome (Pegylated liposome) are under development, with prolonged circulation and residence time in the body. The new developments in the liposome are the specific binding properties of a drug-carrying liposome to a target cell (tumor cell and specific molecules), stealth liposomes for targeting hydrophilic (water soluble) anticancer drugs like doxoru-bicin, mitoxantrone which leads to decrease in side effects because the drug is mostly concentrated at the site of action. Other devel-opment is bisphosphonate-liposome mediated depletion of macro-phages. Several commercial liposomes have already been discov-ered, registered and introduced with great success in pharmaceutical market. There is even greater promise in future for marketing of more sophisticated and highly stabilized liposomal formulations. In future the liposomal drug delivery system will revolutionize the vesicular systems with wide application specially in the treatment of cancer.

REFERENCES

[1] Grubber, S.M. Liposome Biophysics Therapeutics, Marcel Dekker:

New York, 1987.

[2] Bangham, A.D.; Standish, M.M.; Watkens, J.C. J. Mol. Biol., 1965,

13, 238.

[3] Bangham, A.D. Liposomes in Biological Systems, John Wiley and

Sons: Chichester, 1980.

[4] Jayakrishnan, A.; Latha, M.S. Controlled and Novel Drug Delivery,

B.S. Pub: New Delhi, 1997.

[5] Sessa, G.; Weismann, G. J. Biol. Chem., 1970, 245, 3295.

[6] Gregoriadis, G. Methods Enzymol., 1976, 44, 278.

[7] Dunnick, J. K.; Rooke, J D.; Aragon, S.; Kriss, J. P. Cancer. Res.,

1976, 36, 2385-2389.

[8] Hope, M.Y.; Bally, M.B.; Mayer, L.D. Chem. Phys. Lipids., 1986,

40, 89.

[9] Rajendran, D.; Kumari, S.S.; Dhanraj, S.A.; Dube, R.; Suresh, B.

Drug. Dev. Ind. Pharm., 1997, 23(11), 1099.

[10] Freise, J.; Magerstedt, P. Liposomes as Drug Carriers, George

Thieme Verlag: Stuttgart., 1986.

[11] Papahadjopoulos, D.; Nir, S.; Ohki, S. Biochem. Biophys. Acta.,

1971, 266, 561.

[12] Tilcock, C.P.C. Chem. Phys. Lipids, 1986, 40, 109.

[13] Cullis, P.R.; Hope, M.J.; Tilcock, C.P.S. Chem. Phys. Lipids, 1986,

40, 127.

[14] Lasic, D.D.; Frederik, P.M.; Stuart, M.C.A.; Barenholz, Y.; McIn-

tosh, T.J. FEBS Letts., 1992, 312(2, 3), 255.

[15] Ruderman, G.; Gregoriadis, J.R. Biochem. Biophy. Acta, 1986, 863,

277.

[16] Urbaneja, M.A.; Nieva, J.L.; Goni F.M.; Blonso, A. Biochem.

Biophys. Acta, 1987, 904, 337.

[17] Nagarsenker, M.S.; Ashish, A. Drug Dev. Ind. Pharm., 1997,

23(12), 1159.

[18] Ridy, M.S.E.; Khalil, R.M.; Moustafa, D.M.; Rashdy, E.l.; Mo-

hammad, M.; Osman, A.; Gober, M.H.; Monem, A.S. Drug Dev.

Ind. Pharm., 1997, 23(8), 771.

[19] Hwang, K.J.; Padki, M.M.; Chow, D.D. Biochim. Biophys. Acta,

1987, 901, 88.

[20] Papahadjopoulos, D.; Vali, W. J.; Jacobson, K.; Poste, G. Biochim.

Biophys. Acta, 1975, 394, 483.

[21] Pleumchitt, R.; Narong, S.; Korakot, C.; Krisana, K. Drug Dev.

Ind. Pharm., 2000, 29(1), 31.

[22] Mayer, L.D.; Hope, M.; Cullis, P.R. Biochim. Biophys. Acta,

1985b, 817, 193.

[23] Canto, G.S.; Dalmora, S.L.; Oliveira, A.G. Drug Dev. Ind. Pharm.,

1999, 25(12), 1253.

[24] Gregoriadis, G.; Leathwood, P.D.; Ryman, B.E. FEBS Lett., 1971,

19, 95.

[25] Bhalerao, S.S.; Raje Harshal, A. Drug. Dev. Ind. Pharm., 2003, 29,

451.

[26] Liu, D.; Hu, D.; Song, Y. K. Biochim. Biophys. Acta, 1995,

1240(2), 277.

[27] Nakla, A. N.; Szalai, A. J.; Banoub, J. H. J. Liposome Resp., 1994,

4(2), 1029.

[28] Ma, W.; Hwang, K.J.; Lee, V.H.L. Pharm. Res., 1993, 10(2), 252.

[29] Kolchens, S.; Ramaswami, V.; Birgenheier, J.; Nett, L.; O’Brein,

D.F. Chem. Phys. Lipids, 1993, 65, 1.

[30] Talsma, H.; Crommelin, D.J.A. Pharmaceut. Technol., 1992a, 16,

19.

[31] Abra, R.M.; Hunt, C.A. Biochim. Biophys. Acta, 1981, 666, 493.

[32] Jaroni, H.W.; Schubert, R.E.; Schmidt, K.H. Liposomes as Drug

Carries, Georg thieme Verlag: Stutgart, 1986.

[33] Jousma, H.; Talsma, H.; Spies, F. Int. J. Pharm., 1987, 35, 263.

[34] Lasic, D.D. Liposome Biophysics to Application, Elsevier: New

York, 1993.

[35] Lasic, D.D.; Frederik, P.M.; Stuart, M.C.A.; Barenholz, Y.; McIn-

tosh, T.J. FEBS Letts., 1992, 312(2,3), 255.

[36] Lasic, D.D.; Papahadjopoulos, D. Medical Applications of

Liposome, Elsevier: New York, 1998.

[37] Lasic, D.D.; Ceh, D.d.; Stuart, M.C.A.; Guo, L.; Frederik, P.M.;

Barenholtz, Y. Biochim. Biophys. Acta, 1995, 1239, 15.

[38] Lasic, D.D. Liposome in Gene Therapy, CRC press: Boca Raton,

FL, 1997.

[39] Lasic, D.D. Trends Biotechnol., 1998, 16, 307

[40] Lasic D.D. Biochim. J., 1988, 29, 35

[41] Ostro, M. J. Liposome Biophysics to Therapeutics, Marcel Dekker:

New York, 1987.

[42] Mandal, T.K.; Downing, D. T. Acta Derm. Venereol., 1993, 73, 12.

[43] Vyas, S.P.; Katare, Y.K.; Mishra, V.; Sihorkar, V. Int. J. Pharm.,

2000, 210, 1.

[44] New, R.C. Liposomes A Practical Approach, IRL Oxford Univer-

sity Press: Oxford, 1990.

[45] Kolchens, S.; Ramaswami, V.; Birgenheier, J.; Nett, L.; O’Brein,

D.F. Chem. Phys. Lipids, 1993, 65, 1.

[46] Allen, Z.; Tong, X.; Mangeed, P.; Lan, M.; Sydney, U.; Shahid, A.;

Imran, A. Int. J. Pharm., 2004, 270, 93.

[47] New, R.R.C. Liposome A Practical Approach, OIRL press: Oxford,

London, 1989.

[48] Wiener, N.; Lieb, L. Medical Applications of Liposome, Elsevier:

Oxford, 1998.

[49] Weiner, N.; Martin, F.; Riaz, M. Drug Dev. Ind. Pharm., 1989, 15,

1523.

[50] Weiner, N.; Williams, N.; Birch, G.; Ramachandran, C.; Shipman,

C. J. R.; Flynn, G. Antimicrob. Agents Chemother., 1989, 33, 1217.

[51] Chamman, D. Quart. Rev. Biophys., 1975, 8, 185.

[52] Jones, G. R.; Cosins A.R. Liposome A Practical Approach, New

R.R.C Ed, OIRLS press: Oxford, 1989.

[53] Grit, M.; Zuidam, N.J.; Crommelin, D.J.A. Liposome Technology,

CRC press: Boca Raton, FL, 1993.

[54] Chen, C.M.; Alli, D. J. Pharm. Sci., 1987, 76(5), 419.

[55] Uster, P.S.; Deamer, D.W. Arch. Biochim. Biophys., 1981, 209,

385.

[56] Mayer, L.D.; Cullis, P.R.; Balley, M.B. Medical Application of

Liposome, Elsevier science BV: New York, 1998.

[57] Jaroni, H.W.; Schubert, R.E.; Schmidt K.H. Liposomes as Drug

Carries, Georg thieme Verlag: Stutgart, 1986.

[58] Gursoy, A.; Kut, E.; Ozkirimi, S. Int. J. Pharm., 2004, 271, 115.

[59] Fidler, I. J. Science, 1980, 208, 1469.

[60] Fidler, I. J.; Sone, S.; Fogler, W.E.; Barnes, Z. L. Nat. Sci., 1981,

78, 1680.

[61] Deodhar, S.D.; James, K.; Chiang, T.; Edinger, M.; Barna, B.P.

Cancer Res., 1982, 4, 5084.

[62] Thobre, P. S.; Deodhar, S.D. Cancer Immunol. Immunother., 1984,

16, 145.

[63] Poste, G. Liposomes in Biological Systems, John Wiley & Sons,

1980.

[64] Gluck, R.; Mischelar, R.; Brantschen, S.; Just, M.; Althaus, B.;

Cryz, S. J. J. Clin. Invest., 1992, 90, 2491.



[65] De Haan, A.; Tomee, J. F. C.; Huchshorn, J.P.; Wilschut, J. Vac-

cine, 1995, 13, 1320.

[66] Wassef, N.M.; Alving, C.R.; Richards R.L. Immunol. Methods,

1994, 4, 217.

[67] Mc Cauley, J.A.; Flory’s Book.; Mc Comb, T.G. Biochim. Biophys.

Acta, 1992, 30, 112.

[68] Rose, J.K..; Buoncore, L.; Whitt, M.A. Biotechniques, 1991, 10,

520.

[69] Wang, H.S.; Toy, P.C. Biochim. Biophys. Acta, 2002, 15, 25.

[70] Schroeder, U.; Sommerfeld, P.; Ulrich, S.; Sabel, B.A. J. Pharm.

Sci., 1998, 87, 1305.

[71] Alving, C.R.; Steck, E.A.; Chapman, W.L.; Waits, V.B.;

Hendricks, L.D.; Swartz, E.M. Hanson, W.L. Natl. Acad. Sci. USA,

1978, 75, 2959.

[72] Black, C.D.V.; Watson, C.J.; Ward, R.J. Trans. Roy. Soc. Trop.

Med. Hyg., 1977, 71, 550.

[73] Desiderio, J.V.; Campbell, S.G. J. Reticuloendothelial. Soc., 1983,

34, 279.

[74] Desiderio, J.V.; Campbell, S. G. J. Infect. Dis., 1983, 148, 563.

[75] Gabizon, A. Cancer Res., 1992a, 52, 891.

[76] Lawrence, M.; Jennifer, R.M.; Marcel, B. J. Pharm. Sci., 1998,

88(1), 96.

[77] Dinesh, T.; Namdeo, D.; Mishra, P.R.; Khopade, A.J.; Jain, N.K.

Drug Dev. Ind. Pharm., 2000, 23(1), 1315.

[78] Tasuhiro, I.; Yoshihiro, T.; Hisako, D.; Isao, Y.; Hiroshi, K. Int. J.

Pharm., 2002, 232, 59.

[79] Moustapha, H.; Zuzana, H..; Mohamed, A.R.; Susanne, K.; Birgita-

Elfsson, N.K. Cancer Chemother. Pharmacol., 1998, 42, 471.

Received: October 18, 2006 Revised: February 09, 2007 Accepted: February 14, 2007


liposomal drug delivery systems an update review

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