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Chapter

Liposome-A Comprehensive
Approach for Researchers
Mani Sharma, Jyoti Joshi, Neeraj Kumar Chouhan,
Mamta N. Talati, Sandeep Vaidya and Abhiram Kumar

Abstract

Bangham was first to develop these spherical-shaped nano-vesicles called


liposomes in the early 1960s. Today, liposomes have emerged as crucial tools for
bettering the delivery of drugs that majorly includes-antifungal drug, peptide hor-
mones, enzymes, vaccines antimicrobial agents, drugs against cancer, and genetic
materials. Following the different manufacturing practices and versatile properties
liposomes can be categorized in various parameters of size, charge, poly-dispersity
index, encapsulation efficiency, solubility properties, and lamellarity. Alteration in
such parameters elevates the loading and bioavailability of a drug by giving more
clear target specification, desired or controlled release. This bibliographic chapter
provides a comprehensive overview of methods for the preparation of liposomes
with other perspectives that majorly includes—physio-chemical characteristics,
dosage regimen, advantages over other delivery systems, approved liposomal based
drugs and other ongoing drugs in clinical trials. It will help researchers to break-
through more structurally successful delivery vehicles depending upon their various
physic-chemical properties.

Keywords: liposomes, particle size, zeta potential, polydispersity index,


encapsulation efficiency, methods of preparation and bioavailability

1. Introduction

Liposomes can be microscopically examined as the vesicle with spherical


structure that comprises one or more bilayer lipid in the aqueous core part of a
shell. Liposomes are widely used in the delivery of variety of drugs depending upon
its various physic-chemical characteristics. Design and development of liposomes
are classified in many ways among which thin film hydration method is the most
globally accepted procedure. Liposmes formation occurs when lipids are incorpo-
rated into water or buffer solution under continuous stirring, that in return forms
the spherically shaped vesicles termed as liposomes. There are many methods to
develop liposomes among which thin film hydration method is most common.
Recently, lipid film hydration method was used to develop a multilamellar vesicle
(MLV) loaded with curcumin (CUR) and Rhodamine B (RhB), [1] as a successful
drug delivery approach. Phospholipids and cholesterol are the major components
used in the development of liposomes (Figure 1). Where bilayer lipid composes of
a hydrophilic head group, i.e., phospholipid and a hydrophilic tail group. Where
phospholipids can easily penetrate and localize in the skin thus increases the overall

1
Molecular Pharmacology

Figure 1.
Liposome molecule with lipid bilayer.

bioavailability in case of many dermal formulations whereas, cholesterol not only


increases microviscosity of the bilayer but also defines the stability and rigidity of
the formulation [2].
There are many routes to administer liposomes containing drugs, i.e., pulmonary,
ocular, intramuscular, intravenous, topical, nasal and oral. Liposomes can be deliv-
ered in many ways involving sprays, capsules, ointments, creams, solutions, etc. for
curing various disseases: bacterial, fungal, ocular, vaccines, fibrinolysis, endocrine,
arthritis, asthma, diabetes, diseases of immune system, herpes, analgesics, topical
anesthesia and even cancer [3].
Based on different parameters, liposomes are further classified depending
upon method of preparation, structural parameters, biochemistry, cosmetics and
medicine composition, and application in biology. Phospholipids can be from
natural sources such as soya bean, egg yolk and olive oil. Depending upon vari-
ous characteristics liposomes can be categorized on the basis of various physical
parameters such as—pH, temperature, ionic charges, immunogenicity and
stability.
In a recent study performed in 2019, it is revealed that the concentration of
phospholipids and cholesterol variates the protein binding of the formulation [4].
Most commonly employed phospholipids in the formulation of liposomes are:
phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI),
phosphatidylethanolamine (PE), dipalmitoyl phosphatidylserine1,2dioleoylsnglyc
ero3phosphoserine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphati-
dylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) [5].

1.1 Composition of liposomes

A. Phospholipids

1. Derived from natural sources:

• Phosphatidylcholine

• Phosphatidylserine

• Phosphatidylethanolamine

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Liposome-A Comprehensive Approach for Researchers
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2. Synthetic phospholipids:

• Disloyal phosphatidylethanolamine

• Disloyal phosphatidylcholine

B. Cholesterol

Cholesterol are optimized to be used in the formulation of liposomes up to a wide


range with a molar ratios 1:1 or 2:1 against phospholipids. Cholesterol defines a stra-
tegic role in liposome composition; although, the adequate quantity to be used in the
formulation has not been yet clarified. Thus, we can optimize lipids and cholesterol
ratio, to prepare stable and controlled drug release vehicles (Figures 2 and 3) [6].

Figure 2.
Hydrophilic and lipophilic terminals of lipid.

Figure 3.
Inner and outer structure of liposome.

2. Physio-chemical properties

See Tables 1–3.

3. Applications of liposomes

Role of liposome in drug delivery:

• Selective & passive targeting.

• It increases the overall therapeutic index and efficacy of a liposomal


formulation.

3
Molecular Pharmacology

Characterization parameters Analytical method/instrument

Mean vesicle size and size distribution Zetasizer


(submicron and micron range)

Vesicle shape and surface morphology Transmission electron microscopy

Electrical surface potential and surface pH Zetasizer & pH measurement device

Surface charge free Flow electrophoresis

Phase behavior Differential scanning calorimetry (DSC)

Lamellarity Freeze-fracture electron microscopy

Percent of free drug/percent capture Minicolumn centrifugation, ion-exchange


chromatography, radiolabelling

Table 1.
Physical characterization [6].

Characterization parameters Analytical method/instrument

Phospholipid concentration Barlett assay, Stewart assay, HPLC

Concentration of cholesterol By HPLC

Phopholipid peroxidation UV absorbance, iodometric, GLC

Table 2.
Chemical characterization [6].

Characterization parameters Analytical method/instrument

Sterility aerobic or anaerobic cultures Sterility aerobic or anaerobic cultures

Pyrogenicity Limulus amebocyte lysate (LAL) test

Animal toxicity By pathology and histology

Table 3.
Biological characterization [6].

• Due to the encapsulation of drug, overall stability is increased and reduced the
adverse effects of encapsulated drug.

• It helps to improve the pharmacokinetic processes by increasing the circulation


lifetime, decreasing elimination and toxic effects thus elevating the overall
bioavailability of a drug [7].

• Active targeting can also be achieved by coupling with the site-specific


ligands.

3.1 Other advantages of using liposomes

• Biodegradability

• Efficient control of the release

• Resemblance to natural membrane structures

• Increased targeting prospects

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Liposome-A Comprehensive Approach for Researchers
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• Biocompatibility

• Biodegradable

• Liposomes are able to provide both aqueous “milieu internee” and the lipo-
philic environment in a single system

• It helps in protecting the encapsulated drug.

• Method of preparation is easy and has no such complicated or expensive


procedures involved

• Facilitates both active and passive targeting.

• No toxicity in heart as it does not accumulates in the heart.

• Intercepts the oxidation of drug

• Chelation therapy in case of of heavy metal poisoning

• Diagnostic imaging of tumors

• In enzyme replacement therapy

• Study of membranes

• In gene delivery

• As drug delivery carriers

• In multidrug resistance

• In immunology

• In cosmetology (Table 4)

Category Application utilized

In parasitic diseases After IV injection liposomes are comfortly digested by phagocytic cells in the
body and hence considered as one of the best vehicle to dispatch cargo into
macrophages

Anticancer therapy Liposomes are effective for the cells not only in tumors but also in the
gastrointestinal mucosa
Other medical These liposomes are sterically stabilized vesicles and are long circulating micro-
applications reservoirs or tumor (or site of inflammation and infection) targeting vehicles

In bioengineering Fragments of siRNA and DNA are delivered with the help of modern genetic
engineering and gene recombinant technology
In vaccination Liposomes are considerably used in proper vaccination due its fine active targeting

In agro-food industry Due to its versatile physio-chemical properties lipids are extensively manufactured
and used in large scale up sectors

Table 4.
Applications of liposomes [6].

5
Molecular Pharmacology

4. Methods of preparation

See Figure 4.

4.1 Thin film hydration method

This is one of the widely used methods for the preparation of liposomes. As
it has no such complicated steps involved in it. Multilamellar vesicles (MLV) are
prepared by solubilizing natural or synthesized phospholipid in chloroform, dichlo-
romethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v;
2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and
dried in a rota-evaporator under vacuum at a temperature around 45–60°C. Layes is
kept under nitrogen drying for overnight. Next, comes the hydration process where
completely dried thin film is hydrated using aqueous phase—phosphate buffer
solution of pH 7.2 for 1–2 h at 60–70°C.
This kind of procedure can be applied to almost any kind of lipid mixtures, but
has some drawbacks that majorly includes—low encapsulation space, a bit difficult
to scale up and layer formed are not always homogeneous thus shows heterogeneous
size distribution during later physio-chemical examination of liposomes through
zetasizer.

4.2 Injection methods

4.2.1 Ether injection method

Here, the lipid mixture is dissolved in ether or diethyl ether under continuous
stirring that is later injected into a PBS or aqueous phase. Which under injection
pressure causes the removal of almost all organic solvent that ultimately forms
liposomes. This method also suffers with the heterogeneous liposomal formulation
defect.

Figure 4.
General representation for method of preparation of liposome.

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Liposome-A Comprehensive Approach for Researchers
DOI: http://dx.doi.org/10.5772/intechopen.93256

4.2.2 Ethanol injection method

In ethanol injection method the lipid mixture is dissolved in ethanol under contin-
uous stirring that is later injected into a preheated TRIS-HCl buffer or distilled water.
Hydrophobicity and hydrophilicity of a drug accounts for drug intake in a liposomal
vesicle. It has an advantage of using non-toxic and ethanol and is also easily scalable.

4.3 Sonication method

It is the most widely accepted method to develop small unilamellar vesicles


(SLV). SLV are prepared by solubilizing natural or synthesized phospholipid in
chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol
in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this
mixture is revolved and dried in a rota-evaporator under vaccum at a temperature
around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes
the hydration process where completely dried thin film is hydrated using aquous
phase—phosphate buffer solution of pH 7.2 for 1–2 h at 60–70°C. Further the bath
sonicator is used to transform the size of vesicles. Lastly, liposomes are centrifuged
in order to remove the titanium particles that might got added due to overheating in
sonication process. Less encapsulation space is the major drawback of such vesicles.

4.4 High-pressure extrusion method

Liposomes are prepared by solubilizing natural or synthesized phospholipid in


chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol
in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this
mixture is revolved and dried in a rota-evaporator under vacuum at a temperature
around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes
the hydration process where completely dried thin film is hydrated using aqueous
phase—phosphate buffer solution of pH 7.2 for 1–2 h at 60–70°C. In addition, these
lipoosmes are passes through high pressure extruder for 10 cycles in order to obtain
more uniform and stable liposomes.

4.5 Reverse-phase evaporation method

Here, the lipid mixture is dissolved in organic solvents ether or diethyl ether
or a mixture of diethyl ether and chloroform (1:1 v/v); a mixture of methanol-
chloroform (1:2 v/v) under continuous stirring that is later injected into a PBS
or aqueous phase comprising citric-Na2HPO4 to improve the overall efficacy of a
formulation. Which under injection pressure causes the removal of organic solvent
that ultimately leads to the formation of liposomes. This method also suffers with
the heterogeneous liposomal formulation defect. Organic solvent is then dried using
rota-vapor instrument thus forming homogeneous liposome. The major disad-
vantage of this procedure is the leftover of remaining organic solvent in the final
formulation also faces difficulty in scale up procedures.

4.6 Calcium-induced fusion method

Here acidic phospholipids are used to prepare SUV by following the thin film
hydration process followed on with the addition of calcium that causes fusion
to form MLV. Final addition of ethylenediaminetetraacetic acid (EDTA) to MLV
results in the formation of large unilamellar vesicles LUV.

7
Molecular Pharmacology

4.7 Dehydration-rehydration method

Liposomes are prepared by using the sonication method as explained in Section


4.3. Developed liposomes are freeze dried overnight where the formation of multi-
lamellar vesicles occurs when dry powder gets controlled rehydration.

4.8 Freeze-thaw method

Liposomes are prepared by using thin film hydration method as explained in


Section 4.1. Developed liposomes are freeze dried overnight and is then thawed

Drugs liposome formulation Method Type of


liposome
Antifungal drugs
Amphotericin B Thin-film hydration method MLV
Clotrimazole Rotary evaporation method MLV
Fluconazole Thin film hydration method MLV
Analgesic drugs
Ketorolac tromethamine Thin-film hydration method MLV
Antibiotic drugs
Amikacin Reverse phase evaporation method MLV, LUV
Mafenide acetate Solvent evaporation and microencapsulation MLV SUV
Antifibrinolytic drugs
Tranexamic acid Chloroform film and sonication method SUV
Drugs against cancer
5-Fluorouracil Lipid-film hydration method, extrusion, MLV, LUV,
ethanol injection and reverse phase SUV MLV,
evaporation method LUV
Vinblastine sulphate Thin-film hydration method and sonication MLV SUV
Tamoxifen Thin-film hydration method MLV
Bis-demethoxy curcumin analogue Thin-film hydration method and sonication MLV SUV
Doxorubicin Lipid-film hydration method and extrusion MLV
Hormone drugs
Cyproterone acetate Thin-film hydration method MLV
Immunosuppressive drugs
Sirolimus Thin-film hydration method MLV
Tacrolimus (Fk-506) Thin-film hydration method MLV
Ophthalmic drugs
Brimonidine tartrate Thin-film hydration method and sonication MLV SUV
Acetazolamide Reverse phase evaporation and thin-film MLV, LUV
hydration method MLV
Potential drugs as oral insulin
Sodium glycocholate and metformin Reverse phase evaporation and thin-film MLV, LUV
hydrochloride hydration method MLV
Vaccines
Tetanus toxoid diphtheria toxoid Reverse phase evaporation method MLV, LUV

Table 5.
Methods for the preparation of liposomal formulation to deliver drugs [2].

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Liposome-A Comprehensive Approach for Researchers
DOI: http://dx.doi.org/10.5772/intechopen.93256

in order to govern the ionic strength and phospholipid concentration of the final
liposomal formation. Physical disruption of lamellar structure occurs due to freeze-
thaw of liposomal formulation giving it a final ionic structure.

4.9 Microfluidization

Boltic et al. was the first to introduce such method for the preparation of lipo-
somes. Here liposomes are prepared using thin film hydration method as explained
in Section 4.1, which is then sonicated and microfluidized in order to obtain partial
homogenization. This method has its wide application in industrial formulation of
liposomes.

4.10 Supercritical fluids (SCF)

Supercritical fluids (SCF) were introduced to replace toxic organic solvents for
the preparation of liposomes. Supercritical carbon dioxide is the most widely used
supercritical fluid as it has many advantages over conventionally used organic sol-
vents such as—it is not flammable, can be recycled, non-toxic, can be comparatively
easily removed from the solvents, requires moderate temperature and also exclude
the product degradation in inert surroundings. Karn et al. experimented and
explained the comparative study between thin film hydration method and super-
critical fluids using method evaluating the non toxicity and better field approaches
in term of using super critical fluids for the formulation of liposomes (Table 5).

5. Mechanism of liposomal formulation

• Phospholipids shows affinity for polar molecules as well as for aqueous phase
due to a hydrophobic tail, that has 2 fatty acids which are made up of 10–24 C
atoms comprising of 0–6 double bonds in every chain [8].

• In a phospholipid molecule the polar portion connects with a polar environ-


ment of a aqueous medium.

• Phospholipids arrange layers of lipids in close alignment in a planer bilayer


sheet. Sufficient amount of energy is required for this planar arrangement
(sonication, homogenization, heating, etc.) (Figure 5).

6. Evaluation

6.1 Morphological and physicochemical characterization


of liposomal-formulation

The average size, size distribution, and zeta potential shall be determined by
zetasizer.
Transmission electron microscopy is used to study the shape and surface mor-
phology of a liposomal structure.

6.2 In vitro performance evaluation and stability studies

Stability studies: stability studies shall be conducted to assess the shelf-life of


product as per ICH guidelines.

9
Molecular Pharmacology

Figure 5.
Mechanism of liposome formation.

MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to


evaluate the in-vitro cytotoxicity of the developed formulation.
FACS (fluorescence assisted cell sorting) is used to quantify the cell
uptake study.

7. Marketed liposomal formulations

See Tables 6 and 7.

Marketed product Drug used Target diseases Company


AlecTM Dry protein free powder Expanding lung Britannia Pharm, UK
of DPPC PG diseases in babies

VentusTM Prostaglandin E1 Systemic inflammatory The liposome company,


diseases USA
Topex Br Terbutaline sulphate Asthma ozone USA
TM
Doxil or Doxorubicin Kaposi’s sarcoma SEQUUS, USA
CaelyxTM

Novasome Smallpox vaccine Smallpox Novavax USA


TM
Evacet Doxorubicin Metastatic breast The Liposome
Doxorubicin cancer Company, USA

Fungizone® Amphotericin B Fungal infections Leishmaniasis


Depocyt Cytarabine Cancer therapy Skye Pharm USA

Doxil® Doxorubicin HCl Refractory ovarian ALZA, USA


cancer

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Liposome-A Comprehensive Approach for Researchers
DOI: http://dx.doi.org/10.5772/intechopen.93256

Marketed product Drug used Target diseases Company


TM
Amphotec Amphotericin B fungal infections, SEQUUS, USA
leishmaniasis

Table 6.
Liposomal formulations present in the market [9].

Product Manufacturer Liposomes and key ingredients


Formule Liposome Formule Liposome Gel Payot (Thymoxin) hyaluronic acid
Gel (Ferdinand Muehlens)
Symphatic 2000 Biopharm GmbH Thymus extract vitamin A palmitate
Niosomes Lancome (L’Or’eal) Glyceropolyether with moisturizers
Inovita Pharm/Apotheke Thymus extract, hyaluronic
Future Perfect Skin Estee Lauder TMF, Vitamins E, A palmitate,
Gel cerebroside ceramide
Flawless finish Elizabeth Arden Liquid make up
Eye Perfector Avon Soothing cream to reduce eye
Nactosomes Lancome (L’Or’eal) Vitamins
Efect du Soleil L’Or’eal Tanning agents in liposomes niosomes
lancome
Natipide II Nattermann PL Liposomal gel for do-it yourself

Table 7.
Liposomal cosmetic formulations present in the market [10].

8. Conclusions

Liposomes evolved as an extraordinary tool or micro-engineered membranes


for the delivery of drugs because of their minimum toxicity and flexibility that can
be tailored for various desirable intentions. This unparalleled delivery approach
can be used for almost every drug or active pharmaceutical ingredient despite of its
varied physicochemical properties and route of administration. Extensive uses of
liposome in the delivery of drugs can be starched further by researchers, medical
representatives and in scale-up processes in order to develop desired modification
and better delivery approaches by holding the promising physio-chemical proper-
ties and pharmacokinetics (absorption, distribution, metabolism, and elimination)
involved with liposomes, as described in the chapter.

Acknowledgements

I thank all my coauthors who are listed, and the work was not funded by any
institute or person.

Conflict of interest

We wish to declare that there are no known conflicts of interest associated with
this publication, and there has been no significant financial support for this work
that could have influenced its outcome.

11
Molecular Pharmacology

Author details

Mani Sharma1*, Jyoti Joshi1, Neeraj Kumar Chouhan2, Mamta N. Talati2,


Sandeep Vaidya2 and Abhiram Kumar1

1 Uttarakhand Technical University [UKTU], Uttarakhand, India

2 National Institute of Pharmaceutical Education and Research [NIPER],


Telangana, India

*Address all correspondence to: mninup2015@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Liposome-A Comprehensive Approach for Researchers
DOI: http://dx.doi.org/10.5772/intechopen.93256

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