NANOCOCHLEATES: A NOVEL APPROACH FOR DRUG DELIVERY

www.wjpr.net Vol 3, Issue 2, 2014.

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Gol et al. World Journal of Pharmaceutical Research

NANOCOCHLEATES: A NOVEL APPROACH FOR DRUG DELIVERY

Dharmesh Gol*, Viral Shah

Dept. of Pharmaceutics, National Institute of Pharmaceutical Education And Research,

Ahmedabad, Gujarat, India.

ABSTRACT

Nanocochleate is a unique tailor based system used for

microencapsulation and delivery of therapeutics by entrapping them in

supramolecular assemblies composed of negatively charged

phospholipids and a divalent cation. It is a unique multilayered

structure widely used for oral and systemic delivery of wide variety of

molecules including genes, vaccines and antigens. This article

highlights the history, manufacturing methodology, materials used,

stability aspects, characterization, applications and advantages of

nanocochleate drug delivery systems over other vesicular systems. In a

whole nanocochleate represents a unique technology, suitable for oral

and systemic delivery of important chemical and biological

therapeutics and promises a potential drug delivery system

encouraging the future researchers to explore and advance in this new area of delivery

technology.

Keywords: Nanocochleate, liposomes, protein and peptides, vesicular system.

INTRODUCTION

In the past few decades, considerable attention has been focused on the development of new

drug delivery system(NDDS). An ideal NDDS should fulfill two basic requirements:

It should deliver the drug at a rate directed as per the needs of the body, over the period of

treatment and It should channel the active ingredient to the site of action.

The novel drug delivery system is most suitable and approachable in developing the delivery

system which improves the therapeutic efficacy of new as well as pre-existing drugs, thus

providing controlled and sustained drug delivery to the specific site. Conventional dosage

World Journal of Pharmaceutical research

Volume 3, Issue 2, 1920-1944. Review Article ISSN 2277 – 7105

Article Received on 12 December 2013 Revised on 05 January 2013, Accepted on 04 February 2014.

*Correspondence for

Author:

Dharmesh Gol,

Dept. of Pharmaceutics,

National Institute of

Pharmaceutical Education

And Research, Ahmedabad,

Gujarat, India.


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forms are unable to meet any of these requirements. At present, no available drug delivery

system behaves ideally[1]. Now-a-days vesicles as a carrier system have become the vehicle

of choice in drug delivery[2]. Encapsulation of the drug in these vesicular structures is an

system which predicts to prolong the existence of drug in the systemic circulation[3]. The

vesicular systems are highly ordered assemblies of one or several concentric lipid bilayers.

Vesicles consist of a diverse range of amphiphilic building blocks. They can be formed when

these building blocks are confronted with water[4]. Biologic origin of these vesicles was first

reported in 1965 by Bingham, and was given the name Bingham bodies[5]. The ultimate aim

is to control degradation of drug and its loss, prevention of harmful side effects and increase

the availability of the drug at the affected site[6]. For the treatment of intracellular infections,

conventional chemotherapy is considered ineffective, because of limited permeation of drugs

into cells. This can be overcomed by the use of vesicular drug delivery systems[5].

Vesicular drug delivery system has some of the advantages like:

1. Bioavailability is greatly improved, especially in the case of poorly soluble drugs.

2. Both hydrophilic and lipophilic drugs can be incorporated.

3. Delays elimination of rapidly metabolizable drugs and thus eventually function as

sustained release systems[7].

4. Reduces the cost of therapy.

5. It provides an efficient method for drug delivery to the site of infection leading to reduced

drug toxicity[8].

6. Reduces dose-related side effects.

7. Maintains therapeutic concentrations of drugs for longer time duration, thereby

decreasing dosing frequency[7]

TYPES OF VESICULAR SYSTEM

Table No 1: Overview of different vesicular systems along with their chief limitations.

Vesicular system

Description Disadvantages

Liposomes

They consist of one or more concentric lipid bilayers, which enclose an internal aqueous volume[9]

Leakage and fusion of encapsulated drug or molecules, Phospholipids undergoes oxidation and hydrolysis, Short half-life[10]

Niosomes

Drug is encapsulated in an vesicle, which itself is composed of a bilayer of non-ionic surface active agents[11]

Vesicles aggregation, fusion & leaking leading to physical instability, Hydrolysis of medicament[5]


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Pharmacosomes

They are defined as colloidal dispersions of drugs covalently bound to lipids and may exist as ultrafine vesicular, micellar or hexagonal aggregates[12]

Covalent bonding is required to protect the leakage of drugs, Surface and bulk interaction of lipids with drugs is necessary, On storage it undergoes fusion and aggregation leading to hydrolysis[13]

Transferosomes

They are specially optimized, ultraflexible lipid supramolecular aggregates, which consists of inner aqueous compartment surrounded by lipid-bilayers[7]

Chemically unstable due to oxidative degradation, Formulations are expensive, Difficult to maintain purity of natural phospholipids[14]

Cubosomes

They are discrete, sub micron, nanostructured particles of bicontinuous cubic liquid crystalline phase[15]

Difficult to manufacture them on a large scale, because of low viscosity[16]

Sphingosomes

It is bilayered vesicles in which an aqueous volume is entirely enclosed by membrane lipid bilayer mainly composed of natural or synthetic sphingolipid.

Low entrapment efficacy, Higher cost of sphingolipid hinders its preparation & use.

Colloidosomes

They are hollow shell microcapsules consisting of coagulated or fused particles at interface of emulsion droplets[7]

Poor yield of particles, If shell locking is inefficient, they simply coalesce & fall apart. [18]

Ethosomes They are lipid “Soft malleable-vesicles” embodying a permeation enhancer. Generally composed of phospholipid, ethanol and water[19]

Poor yield, Coalesce & fall apart if shell locking is inefficient, Loss of contents during transfer from organic to water media[20]

Discosomes These are disc-shaped niosomes solublized with non-ionic surfactant solutions(Poly-Oxy-Ethylene Cetyl Ether class). Ligand mediated drug targeting is shown by them[21]

High cost of preparation, Poor yield.

LIPOSOMES

vesicle-based delivery systems particularly liposomes were quite successful in delivery of

drugs across the membrane, because of their structural similarity with the cell membrane.

Liposomes were first described by British haematologist, Dr. Alec Bangham in 1961 at the

Babraham Institute in Cambridge. Liposomes were discovered when Bangham and R. W.

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

phospholipids. Liposomes resembled to the structure of plasma membrane. From the

microscopic images, first evidence was obtained for the bilayer lipid structure of cell-

membrane. The word liposome was derived from two Greek words: lipo means "fat" and

soma means "body"; it is so named because its composition is primarily of phospholipid[22,


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23]. Liposomes are considered to be simple microscopic vesicles in which an aqueous volume

is entirely enclosed by a membrane composed of lipid molecule.”[24].

Fig. 1: Basic liposome structure.

Advantages of liposomal drug delivery system:

Provides selective passive targeting to tumor tissues, increases efficacy and therapeutic index

of drug molecule, increases stability via encapsulation, reduces toxicity of the encapsulated

agents, shows site avoidance effect, improves pharmacokinetic parameters of drug molecule

(reduced elimination, increased circulation life times), imparts flexibility to couple with site

specific ligands to achieve active targeting[25], help to reduce exposure of sensitive tissues to

toxic drugs[24], liposomes are biocompatible, completely biodegradable, non-toxic, flexible

and non-immunogenic for systemic and non-systemic administrations.

Types of liposomes[26-31]

Liposomes are classified on the basis of their structural parameters, method of preparation,

composition and applications.

[a] Based on structural parameters

1. Uni-Lamellar Vesicles:

Small Uni-lamellar Vesicles(SUV):Size ranges from 20-40 nm

Medium Uni-lamellar Vesicles(MUV):Size ranges from 40-80 nm.

Large Uni-lamellar Vesicles(LUV):Size ranges from 100-1000 nm.

2. Oligo-Lamellar Vesicles(OLV): These are made of 2 to 10 lipid bilayers surrounding a

large internal volume.


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3. Multi-Lamellar Vesicles(MLV):They compose of several lipid-bilayers. Their

arrangement can be ONION-like concentric spherical bilayers of LUV/MLV enclosing a

large number of SUV etc.

[b] Based on method of liposome preparation

1. REV:- Single or Oligolamellar vesicles made by Reverse-Phase Evaporation Method.

2. MLV-REV:- Multi-lamellar Vesicles made by the Reverse-Phase Evaporation Method.

3. SPLV:- Stable Pluri-Lamellar Vesicles.

4. FATMLV:- Frozen & Thawed MLV.

5. VET:- Vesices prepared by Extrusion Technique.

6. DRV:- Dehydration-Rehydration Method.

7. BSV:- Bubblesomes.

[c] Based on composition & application

1. Conventional Liposomes(CL):- Neutral or negatively charged phospholipids &

cholesterol.

2. Fusogenic Liposomes(RSEV):- Reconstituted Sendai Virus Envelopes.

3. pH sensitive Liposomes:- Phospholipids such as phosphatidyl-ethanolamine(PE) or 1,2

dioleoyl phosphatidyl-ethanolamine(DOPE).

4. Cationic Liposomes:- Cationic lipids with DOPE.

5. Long Circulatory(Stealth) Liposomes(LCL):- They have Poly-Ethylene Glycol(PEG)

derivatives attached to their surface to decrease their detection by phagocyte system. The

attachment of PEG to liposomes decreases the clearance from blood stream & extends

circulation time of liposomes in the body. The attachment of PEG is also known as

“Pegylation”.

6. Immuno-Liposomes:- CL or LCL with attached monoclonal antibody or recognition

sequence.

Architect of liposomes[32]

1. Phospholipids: Glycerol represents more than 50% of the weight of lipid in the

biological membranes. Thus glycerol containing phospholipids are most common in use.

These are derived from phosphatidic acid. For stable liposomes, saturated fatty acids are

generally used, whereas unsaturated fatty acids are less common in use. Examples of

phospholipids are:-phosphatidyl Choline(Lecithin)(PC), phosphatidyl Serine(PS),

phosphatidyl Glycerol(PG), phosphatidyl Inositol(PI).


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2. Sphingolipids: They are present in animal & plant cells. They contain three characteristic

building blocks, fatty acid molecule, sphingosine molecule and a head group (simple alcohol,

choline or complex carbohydrates). Most common sphingolipids are sphingomyelin &

glycosphingo lipids.

3. Sterols: Cholesterol & its derivatives are used in liposome preparation. They serve

following functions in liposomes, decreasing fluidity, reducing permeability of membrane to

water soluble molecules and stabilizing membrane in presence of biological fluids. In the

absence of cholesterol, liposomes tend to interact with plasma proteins. These proteins extract

the phospholipids from liposomes, thereby depleting the outer monolayer of vesicles creating

physical instability. Cholesterol reduces this type of interaction.

4. Synthetic phospholipids

Examples of saturated phospholipids are dipalmitoyl phosphatidyl choline(DPPC), distearoyl

phosphatidyl choline(DSPC), dipalmitoyl phosphatidic acid(DPPA), dipalmitoyl

phosphatidyl glycerol(DPPG).

Examples of unsaturated phospholipids are, dioleoyl phosphatidyl choline(DOPC), dioleoyl

phosphatidyl glycerol(DOPG).

5. Polymeric materials: Synthetic phospholipids containing diacetylenic group in the

hydrocarbon chain when exposed to U.V. light polymerizes & leads to formation of

polymerized liposomes. These liposomes have high permeability barriers to entrapped drugs.

6. Polymer bearing lipids: Stability of repulsive interactions is governed by repulsive

electrostatic forces. This repulsion can be induced by coating surface of liposome with

charged polymers. Non-ionic & water compatible polymers like polyethylene oxide,

polyvinyl alcohol & poly-oxa-zolidines can be used for coating liposomal surface.

7. Cationic lipids

DODAB/C- Di-Octa-Decyl Dimethyl Ammonium Bromide/Chloride.

DOTAP- Di-Oleoyl Propyl Trimethyl Ammonium Chloride.


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Methods of liposome formation

Fig. 2: Mechanism of liposome formation[33]. Fig. 3: General scheme for liposome

preparation & drug loading[34]

Fig. 4: Methods of liposome preparation[34].

Therapeutic applications of liposomes[24]

a) Liposome are used for drug/protein delivery vehicles: For controlled and sustained drug

release, enhanced drug solubilization, altered pharmacokinetics and biodistribution, enzyme

replacement therapy and biodistribution, enzyme replacement therapy and lysosomal storage

disorders.


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b) Liposome in tumor therapy is used as a carrier of small cytotoxic molecules & vehicle for

macromolecules as cytokines or genes.

c) Liposomes are used in Gene Delivery for gene and antisense therapy as well as genetic

(DNA) vaccination.

d) Liposome are used as immuno-adjuvants, immuno-modulator & in immuno-diagnosis.

e) Liposome are also used as artificial blood surrogates.

f) Liposome are used as radio diagnostic carriers.

g) Liposome are used in several cosmetics and dermatology products.

h) Liposome is also used in enzyme immobilization and bioreactor technology.

Limitations of liposomal drug delivery[24]

Cost of production is high, phospholipid may sometimes undergo oxidation & hydrolysis like

reaction, short half-life, low solubility, poor mechanical stability due to leakage & fusion of

the formulation, low entrapment efficiency.So the need of the hour was to develop an

formulation to answer the above limitations of liposome based vesicular drug delivery

systems. Cochleates are the vesicular system which could satisfy the present needs of the

market.

COCHLEATE: Various formulation modifications with liposomes allowed the development

of a new class of drug vehicles called “COCHLEATE”[35]. Cochleates are solid particulates

made of large continuous, lipid bi‐layer sheets rolled up in a spiral structure with no internal

aqueous phase. This technology was able to answer the challenges of oral delivery of

different kind of biological molecules, especially the hydrophobic ones. Cochleates differ

from liposomes in having water-free interior, rod-shape & rigid stable structure[36].

Fig. 5: Structural difference between liposomes & cochleates.


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These unique characteristics make cochleates a great platform for delivery of drugs that were

having poor bioavailability[36]. It is most versatile technology for the delivery of a wide range

of drugs and molecules such as proteins and peptides, polynucleotide, antiviral agent,

anaesthetic, anticancer agent, immunosuppressant, steroidal anti-inflammatory agent, non-

steroidal anti-inflammatory agents, tranquilizer, nutritional supplement, herbal product,

vitamin. Thus it provides a potential delivery system for the wide class of drugs[37].

History: Cochleates were discovered in 1975 by Dr. Dimitrious Papahadjoupoulos and his

co‐workers as precipitates formed by the interaction of negatively charged phosphatidyl-

serine and calcium. He named these cylindrical structures "COCHLEATE". In Greek, it

means “SHELL” because of their rolled-up form[38]. In the late ‘80s & ‘90s, cochleate were

used to transport antigens and peptides for vaccine delivery. Cochleate structure is either

aggregates of stacked sheets formed by trapping method or large size needles‐like structures

formed by the dialysis method[39]. In 1999, cochleates were introduced to develop smaller,

but rather more consistent particles. It was demonstrated that by using a binary phase system,

such as two non‐miscible hydrogels; cochleates can be formed that display a small mean

particle of less than 500 nm. These nanocochleates were highly suitable for the encapsulation

of hydrophobic drugs[40].

In the initial time, cochleates were prepared in micrometer sizes[41] either by direct addition

of multivalent ion solution to liposome’s solution or by the dialysis method. However, the

particle size could not be reduced to nanometer range. Perhaps in recent past a new method

named “Hydrogel-Isolated cochleation” to prepare nanometer-sized cochleates was coined[42].

Basics: Cochleate and nanocochleate are cigar like spiral rolls formed of negatively charged

phospholipid bilayers, which are rolled up through the interaction with multivalent counter

ions(Ca2+ or Zn2+) as bridging agents between the bilayers. They roll-up in order to minimize

their interaction with water[39]. They possess little or no aqueous phase. The entire

nanocochleate structure is a series of solid layers. Thus even if the outer layers of

nanocochleate are exposed to harsh environmental conditions or enzymes, the encapsulated

drug molecules will remain intact within the interior[43].As a particulate system, cochleates

possess unique properties like superior mechanical stability and better protection for

encapsulated drugs compared with liposomes due to their solid matrix. These solid particles

are so flexible that they can readily convert to liposomes by extracting the bridging counter


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ions out of the inter bilayer spaces[44]. Nanocochleates contain both hydrophobic and

hydrophilic surface which makes it suitable for encapsulation of both hydrophobic drugs like

amphotericin B and clofazimine and amphiphilic drug like doxorubicin. The loading capacity

of the cochleates depends upon the physical chemistry of the drug to encapsulate, whereas the

particle size of the complex formed depends on the process used to encapsulate[36]. Such

unique properties have made cochleates an ideal system for delivering insoluble ingredients

which can be loaded in the matrix of a phospholipid bilayer while avoiding the instability

problem of liposomes [44].

Fig. 6: Nanocochleate formation by interaction between negative lipids and cations[45].

Components of nano-cochleate drug delivery system[46]:

The three major components used in preparation of nanocochleates are:-API, Lipids and

Cations.

Table No 2: Components of nanocochleate drug delivery system

LIPIDS

Phosphatidyl Serine[PS], Phosphatidic Acid[PA], Di-Oleoyl Phosphatidyl

Serine[DOPS], Phosphatidyl Inositol[PI], Phosphatidyl Glycerol[PG],

Phosphatidyl Choline[PC], Di-Myristoyl Phosphatidyl Serine[DMPS],

Phosphatidyl Ethanolamine[PE], Di-Phosphatidyl Glycerol[DPG], Dioleoyl

Phosphatidic Acid (DOPA), Di-Stearoyl Phosphatidyl Serine[DSPS], Di-

Palmitoyl Phosphatidyl Gycerol[DPPG].

CATIONS Zn+2 or Ca+2 or Mg+2 or Ba+2


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Table No 3: Brief description for some lipids used in nanocochleate delivery systems

Lipids Description

Phosphatidyl

choline(PC)

It is the major component of lecithin. It is the main functional constituent of

the natural surfactants and the body's foremost reservoir of choline, an

essential nutrient[47].

Phosphatidyl

ethanolamine

(PE)

1,2-Diacyl-glycero-3-phospahtidyl ethanolamine is the second most

abundant phospholipid in animal and plant lipids. It is the main lipid

component of microbial membranes. It is a key building block of membrane

bilayers[48].

Phosphatidyl

inositol (PI)

It is an important lipid as it is a key membrane constituent. Also it is an

participant in essential metabolic processes in all plants and animals and in

some bacteria [49].

Di-

phosphatidyl

glycerol(DPG)

Also named as Cardiolipin. It is found exclusively in certain membranes of

bacteria(plasma membrane & hydrogenosomes) and mitochondria of

eukaryotes.

Phosphatidyl

glycerol(PG)

It is a constituent of cell membranes typically present at 1-2% concentrations

in most animal tissues. It is an important precursor of cardiolipin. It is found

in all bacteria types.

Methods of nanocochleate preparation: Nanocochleates are derived from liposomes which

are suspended in an aqueous two-phase polymer solution, enabling the differential

partitioning of polar molecule based-structures by phase separation. The liposome-containing

two-phase polymer solution, treated with positively charged molecules such as Ca++ or

Zn++, forms a nanocochleate precipitate of a particle size less than one micron. The process

may be used to produce nanocochleates containing biologically relevant molecules[50].There

are several methods for cochleate preparation:-

1.Hydrogel method: This method comprises of following steps:

Step1: A suspension of small unilamellar liposomes or biologically relevant molecule-loaded

liposomes is prepared. This can be achieved by standard methods such as sonication or

microfluidization or other related methods.

Step2:The liposome suspension is mixed with polymer A such as dextran (mol wt-200,000-

500,000), Polyethylene glycol (mol wt- 3400-8000) or Phosphatidylserine.


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Step3:Preferably by injection, the liposome/Polymer A suspension is added into polymer B

such as poly vinyl pyrrolidone, poly vinyl alcohol, ficoll (mol wt- 30,000-50,000), and poly

vinyl methyl ether (PVMB)(mol wt- 60,000-160,000) in which polymer A is not miscible,

leading to an aqueous two-phase system of polymers. This can be achieved mechanically by

using a syringe pump at an appropriate controlled rate, for example a rate of 0.1 ml/min to 50

ml/min, and preferably at a rate of 1 to 10 ml/min.

Step4:A solution of cation salt is added to the two-phase system of above step, such that the

cation diffuses into polymer B and then into the particles comprised of liposome/polymer A,

allowing the formation of small-sized cochleates.

Step5:To isolate the cochleate structures and to remove the polymer solution, cochleate

precipitates are repeatedly washed with a buffer containing a positively charged molecule,

and more preferably, a divalent cation. Addition of a positively charged molecule to the wash

buffer ensures that the cochleate structures are maintained throughout the wash step, and that

they remain as precipitates(fig:7)[51].

Fig. 7: Hydrogel isolation method[42].

2.Liposome before cochleates(LC)/dialysis method: In this method mixture of lipid and

detergent are used as the starting material and the removal of detergent is made by double

dialysis[52]. The detergent is added to disrupt the liposomes. The method comprises the


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following steps: Step1: An aqueous suspension containing a detergent-lipid mixture is

prepared.

Step2:The detergent-lipid suspension is mixed with polymer A such as dextran (mol wt-

200,000-500,000), Polyethylene glycol (mol wt- 3400-8000) or Phosphatidyl-Serine.

Step3:The detergent-lipid/polymer A suspension is added into a solution comprising polymer

B such as poly vinyl pyrolidone, poly vinyl alcohol, ficoll (mol wt- 30,000- 50,000), and poly

vinyl methyl ether (PVMB) (mol wt- 60,000- 160,000), wherein polymer A and polymer B

are immiscible, thereby creating a two-phase polymer system.

Step4:A solution of a cationic moiety is added to the two-phase polymer system.

Step5:Washing the two-phase polymer system to remove the polymer(fig.8)[51]

3. Direct calcium(DC) dialysis method: Unlike LC method this method dose not involves

the intermediate liposome formation and the cochleates formed were large in size. The

mixture of lipid and detergent was directly dialyzed against calcium chloride solution. In this

method a competition between the removal of detergent from the detergent/lipid/drug

micelles and the condensation of bilayers by calcium, results in needle shaped large

dimensional structures.

Step1: Mixture of phosphatidylserine and cholesterol (9:1 wt ratio) in extraction buffer and

non‐ionic detergent was mixed with a preselected concentration of API and the solution was

vortexed for 5 minutes.

Step2: The clear, colorless solution was then dialyzed at room temperature against three

buffers.

Step3: The final dialysis is done in 6 mM Ca2+ solution and buffer concentrations are

maintained compatible to cochleate formation. The resulting white calcium‐phospholipid is

DC cochleate[50].

4.Binary aqueous-aqueous emulsion system: In this method small liposomes were formed

by either high pH or by film method, and then the liposomes are mixed with a polymer, such

as dextran. The dextran/liposome phase is then injected into a second, non-miscible, polymer

(i.e. PEG). The calcium was then added and diffused slowly from one phase to another

forming nanocochleates. By this method the cochleates formed are of particle size less than

1000 nm(fig.9)[50].


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5. Trapping method: This method is useful for the encapsulation of hydrophilic and

hydrophobic molecules[39, 52]. It consist of preparation of the liposomal suspension containing

the drug either in the aqueous space of liposome (when hydrophilic) or intercalated in

between the bilayers(when hydrophobic). A step of addition of calcium follows, and an

aggregate of cochleates are formed. The cochleates made by the trapping method present

higher aggregation compared with other methods. This has been demonstrated using electron

microscopy after freeze-fracture(fig.10)[53].

6. Solvent drip method: It consists of preparing a liposomal suspension separately based on

soy PS and a hydrophobic or amphipathic cargo moiety solution. Solvent for hydrophobic

drug can be selected from DMSO or DMF. The solution is then added to liposomal

suspension. Since the solvent is miscible in water, a decrease of the solubility of the cargo

moiety is observed, which associates at least in part with the lipid-hydrophobic liposomal

bilayers. The cochleates are then obtained by addition of calcium and the excess solvent is

being washed[54].

Route of administration

Nanocochleate drug delivery vehicle allows an efficient oral delivery of drugs. An alternative

route of administration can be parentral, rectal, topical, sublingual, mucosal, nasal, opthalmic,

subcutaneous, intramuscular, intravenous, transdermal, spinal, intrathecal, intra-articular,

intra-arterial, sub-arachnoid, bronchial, lymphatic, and intrauterine administration,

intravaginal or any other mucosal surfaces[42].

Dosage forms for nanocochleate administration: For oral administration: capsules,

cachets, pills, tablets, lozenges, powders, granules, or as a solution or a suspension or an

emulsion, for topical or transdermal administration: powders, sprays, ointments, pastes,

creams, lotions, gels, solutions, patches and inhalants, for parentral administration: sterile

isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile

powders which may be reconstituted into sterile injectable solutions or dispersions just prior

to use[55].

Stability of nanocochleate formulations

Encapsulation of the drug molecules in the interior provides protection and stability to the

entire formulation. Because the entire structure is a series of solid lipid bilayers, components

within the interior of this structure remain intact, even though the outer layers of it may be


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exposed to harsh external environmental conditions or enzymes. The interior is essentially

free of water and resistant to penetration by oxygen which has been resulted into increased

shelf-life of the formulation. Nanocochleates may be lyophilized to a powder and stored at

room temperature or 40°C. Lyophilized cochleates can be reconstituted with liquid prior to

in-vitro use or in-vivo administration. Lyophilization has no adverse effects on cochleate’s

morphology or functions[54].

Safety & compatibility of nano-cochleate formulations

Phosphatidyl-Serine(PS) and calcium which are safe, simple, naturally occurring substances

which makes nanocochleate a safe and biocompatible delivery vehicle as its an natural

component of all biological membranes concentrated mostly in the brain. Soyabean

Phosphatidyl-Serine is also an alternative source & its inexpensive, available in large

quantities and suitable for use in humans. Nanocochleates which are composed of anionic

lipids are non-inflammatory and bio-degradable[54].

Mechanism of drug delivery through cochleate formulation: Absorption after oral

administration takes place from intestine. Nanocochleates cross across the digestive

epithelium and deliver their active drug molecules into blood vessel. In case of other route

(except IV) they cross across the associated cell and reach into circulation. After reaching

into circulation they are delivered to targeted cell(fig.11)[50].

Fig. 8: Absorption mechanism of nanocochleate

The recently proposed hypothesis states that when lipid bi-layer structure of nanocochleates

fuses with the cell membrane, then the contents of nanocochleates are delivered into cells,

thus release of the drug occurs(fig.12)[36].


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Fig. 9: Direct interaction and cell membrane fusion of nanocochleate formulation[36]

Nanocochleate first comes into close approximation to a natural membrane, a perturbation

and reordering of the cell membrane is induced, resulting in a fusion event between the outer

layer of the nanocochleate and the cell membrane. This fusion results in the delivery of a

small amount of the encochleated material into the cytoplasm of the target cell. The

nanocochleate may slowly fuse or break free of the cell and be available for another fusion

event, either with this or another cell[50].

ADVANTAGES: Below are few advantages of nano-cochleate formulation over the other

delivery systems. Nanocochleates have a non-aqueous structure and therefore:

1. They are more stable than liposomes because the lipids in nanocochleates are less

susceptible to oxidation. They maintain structure even after lyophilization, whereas

liposome structures are destroyed by lyophilization[50].

2. With administration of live vaccine, there are many life threatening risk associated like

allergies, inversion of vaccine effect to wild infection etc. These can be neglected by use

of nanocochleates.

3. Multiple administrations of high doses of cochleate formulations to the same animal shows

no toxicity and do not result in either the development of an immune response to the

cochleate formulation.

4. They exhibit efficient incorporation of biological molecules, particularly with hydrophobic

moieties into the lipid bilayer of the cochleate structure.

5. They have the potential for slow or timed release of the biological molecule in-vivo as

nanocochleates slowly unwind or otherwise dissociate.

6. They have a lipid bilayer matrix which serves as a carrier and is composed of simple lipids

which are found in animal and plant cell membranes, so that the lipids are non-toxic, non-

immunogenic and non-inflammatory.


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7. They improve oral bioavailability of a broad spectrum of compounds, such as those with

poor water solubility, protein and peptides etc.

8. They reduce toxicity, stomach irritation and other side effects of the encapsulated drug.

9. They encapsulate or entrap the subject drug within a crystal matrix rather than chemically

bonding with the drug.

10. They provide protection from degradation to the encochleated drug caused by exposure to

environmental conditions such as sunlight, oxygen, water and temperature.

CHARACTERIZATION OF NANO-COCHLEATE FORMULATION

Particle size determination: The mean particle size of the liposomal dispersion and

cochleates dispersion can be determined by laser diffraction technique using Malvern

analyzer. Analysis is to be carried out at 30±2°C temperature keeping angle of detection

90°C[56].

Entrapment efficiency(EE) : One hundred micro liters of cochleates is aliquoted into

centrifugation tubes. To each tube 60 µl pH 9.5 EDTA and 1ml of ethanol is added while

vortexing. Absorbance of the resulting solution is determined using spectroscopic technique

and entrapment efficiency is calculated using below mentioned equation[56].

Stability study: Cochleates dispersions can be kept at 2-8°C and 25±2°C/60% RH for a

period of 3 months to check their stability. The stability of the vesicles is determined in terms

of change in particle size and percent entrapment efficiency[56].

Cochleates-cell interaction: To examine the interaction of cochleates with cell membrane,

2% fluorescent lipid in addition to the negatively charged lipid is used to form fluorescent

liposomes[57, 58]. When cochleates interact with cell membrane involving a fluorescent lipid

transfer, cell surfaces become fluorescent under fluorescent microscopes as illustrated in fig.

13-A and B


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[41].

Fig. 10-A & B Mammalian skin cells exposed to the poly-lysine fluorescent

nanocochleate.

Specific surface area: The specific surface area of freeze-dried nanocochleate can be

determined with the help of a sorptometer. The equation given below can be used to calculate

specific surface

Here A is the specific surface area, ρ is the density and d is the diameter of the cochleate[41].

Surface charge: The nature and intensity of the surface charge of nanocochleate determines

their interactions with the biological environment as well as their electrostatic interaction

with bioactive compounds. The surface charge can be determined by measuring the particle

velocity in an electric field. Laser light scattering techniques such as Laser Doppler

Anemometry or Velocimetry (LDA/LDV) are used as fast and high-resolution techniques for

determining nanocochleate velocities. The surface charge of colloidal particles can also be

measured as electrophoretic mobility. The composition of charge decides the bio-distribution

of drug carrying nanocochleate. Generally, the electrophoretic mobility of nanocochleate is

determined in a phosphate saline buffer and human serum. The phosphate saline buffer (pH

7.4) reduces the absolute charge value due to ionic interaction of buffer components with the

charged surface of nanocochleate. [58]

In vitro release study: The in vitro release profile of nanocochleates is determined using

standard dialysis, diffusion cell or modified ultra-filtration techniques. Phosphate buffer with


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double chamber diffusion cells on a shake stand is generally used. A Millipore, low protein-

binding membrane is placed between the two chambers. The donor chamber is filled with

nanocochleates and the receptor compartment is assayed at different time intervals for the

released drug using standard procedures. The modified ultra-filtration technique is also used

to determine the in vitro release behavior of nanocochleates. Here nanocochleate is added

directly into a stirred ultra-filtration cell containing buffer. At different time intervals,

aliquots of the medium are filtered through the ultra-filtration membrane & assayed for the

released drug using standard procedures[59].

APPLICATIONS

1. For delivery of antibiotics:- Cochleate delivery system has potential for the delivery of

antibacterial agents such as Aminoglycosides and Anti-TB drugs[60, 61]. As to illustrate the

Clofazimine entrapped cochleates exhibit a greater decrease in toxicity versus free

clofazimine and had a higher efficacy in killing intracellular M. Tuberculosis than free

clofazimine. This shows that encapsulation of clofazimine in cochleates potentiates the

antimicrobial efficacy of the drug [62].

2. As carriers for topical delivery:- This novel structure reported to be more stable than

liposomes, aids in dermal and transdermal release of the active constituent. Ketoconazole

loaded cochleates retained antifungal activity with better permeability across the skin[56].

3. Nanocochleates can provide a good platform for the delivery of formulations by oral

administration and can bring a revolution in the treatment of atherosclerosis and heart

diseases originating from high blood cholesterol and LDL levels.

4. Nanocochleates have the ability to stabilize and protect an extended range of

micronutrients and the potential to increase the nutritional value of processed foods.

5. Nanocochleates have been used to deliver proteins, peptides and DNA for vaccine and

gene therapy applications.

6. Nanocochleates showed potential to deliver Amphotericin B, a potential antifungal agent,

orally and parentally having a good safety profile with reduced cost of treatment. The

prepared cochleates of amphotericin B showed improved stability and efficacy at low doses.

They showed improved patient compliance & the formulation was approved by USFDA.

7. Bio Delivery Sciences International(BDSI), an US based company have developed

nanocochleates which can be used to deliver nutrients such as vitamins, omega fatty acids

more efficiently to cells, and lycopene without affecting the color and taste of food which

makes the concept of super foodstuffs a reality [35, 63-65].


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CONCLUSION

Nano-cochleate drug delivery system provides an unique platform for delivery of wide range

of oral & systemic therapeutics including drugs, genes, and vaccine antigens. Encochleation

helps to improve the efficiency of the final product by enhancing the qualities of formulation,

increasing processing and shelf-life stability, enhancing bioavailability, reducing toxicity, and

increasing efficacy. As nanocochleate possesses unique multilayered structure, it protects

active agents inside which are to be carried. Thus nanocochleates defeats the disadvantages of

other drug delivery systems. There is tremendous increase in patent filing and publications of

nanocochleates indicating growing industrial interest as well as academic interest in the area

of drug delivery. Thus nanocochleate drug delivery system is gaining more importance in

pharmaceutical development for transfer of suitable & desired drug molecule into body with

good potential.

ACKNOWLEDGEMENT

The authors are thankful to Project Director NIPER-Ahmedabad and to Director of mentor

Institute, Shri. B.V. Patel PERD centre Ahmedabad, for constant support and motivation

during the write up of the manuscript whose inhouse manuscript no. is NIPERA380114.

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NANOCOCHLEATES: A NOVEL APPROACH FOR DRUG DELIVERY