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Chapter I Introduction

Formulation Development and In vivo Evaluation of Zidovudine Niosomes

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CHAPTER - I

INTRODUCTION

1.1 NOVEL DRUG DELIVERY SYSTEM (NDDS)

The basic goal of novel drug delivery system (Remington, 2001) is to

achieve a steady state blood or tissue level that is therapeutically effective and

non toxic for an extended period of time.

Conventional drug delivery involves the formulation of the drug into a

suitable form, such as compressed tablet for oral administration or a solution for

IV administration. These dosage forms have been found to have serious

limitations in terms of higher doses required lower effectiveness, toxicity and

adverse effects. NDDS are being developed rapidly, so as to overcome the

limitations of conventional drug delivery.

The method by which a drug is delivered can have a significant effect on

its efficacy (Costas Kaparissides et al., 2006). Some drugs have an optimum

concentration range within which maximum benefit is derived, and concentrations

above or below this range can be toxic or produce no therapeutic benefit at all.

On the other hand, the very slow progress in the efficacy of the treatment of

severe diseases, has suggested a growing need for a multidisciplinary approach

to the delivery of therapeutics to targets in tissues. From this, new ideas on

controlling the pharmacokinetics, pharmacodynamics, non-specific toxicity,

immunogenicity, biorecognition, and efficacy of drugs were generated.

These new strategies, often called drug delivery systems (DDS), are based on

interdisciplinary approaches that combine polymer science, pharmaceutics,

bioconjugate chemistry, and molecular biology.

To minimize drug degradation and loss, to prevent harmful side-effects

and to increase drug bioavailability and the fraction of the drug accumulated in

the required zone, various drug delivery and drug targeting systems are currently

under development. Among drug carriers one can name soluble polymers, micro



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particles made of insoluble or biodegradable natural and synthetic polymers,

microcapsules, cells, cell ghosts, lipoproteins, liposomes, niosomes and micelles.

The carriers can be made slowly degradable, stimuli-reactive (e.g., pH- or

temperature-sensitive), and even targeted (e.g., by conjugating them with specific

antibodies against certain characteristic components of the area of interest).

Targeting is the ability to direct the drug-loaded system to the site of interest. Two

major mechanisms can be distinguished for addressing the desired sites for drug

release: (i) passive and (ii) active targeting. An example of passive targeting is

the preferential accumulation of chemotherapeutic agents in solid tumours as a

result of the enhanced vascular permeability of tumor tissues compared with

healthy tissue. A strategy that could allow active targeting involves the surface

functionalization of drug carriers with ligands that are selectively recognized by

receptors on the surface of the cells of interest. Since ligand–receptor interactions

can be highly selective, this could allow a more precise targeting of the site of

interest.

Controlled drug release and subsequent biodegradation are important for

developing successful formulations. Potential release mechanisms involve: (i)

desorption of surface-bound /adsorbed drugs; (ii) diffusion through the carrier

matrix; (iii) diffusion (in the case of nanocapsules) through the carrier wall; (iv)

carrier matrix erosion; and (v) a combined erosion/diffusion process. The mode of

delivery can be the difference between a drug’s success and failure, as the

choice of a drug is often influenced by the way the medicine is administered.

Sustained (or continuous) release of a drug involves polymers that release the

drug at a controlled rate due to diffusion out of the polymer or by degradation of

the polymer over time. Pulsatile release is often the preferred method of drug

delivery, as it closely mimics the way by which the body naturally produces

hormones such as insulin. It is achieved by using drug-carrying polymers that

respond to specific stimuli (e.g., exposure to light, changes in pH or temperature).

For over 20 years, researchers have appreciated the potential benefits of

nanotechnology in providing vast improvements in drug delivery and drug

targeting. Improving delivery techniques that minimize toxicity and improve



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efficacy offers great potential benefits to patients, and opens up new markets for

pharmaceutical and drug delivery companies. Other approaches to drug delivery

are focused on crossing particular physical barriers, such as the blood brain

barrier, in order to better target the drug and improve its effectiveness; or on

finding alternative and acceptable routes for the delivery of protein drugs other

than via the gastro-intestinal tract, where degradation can occur.

1.2 ADVANTAGES OF NOVEL DRUG DELIVERY SYSTEM

1) Reduce the number and frequency of doses required to maintain the

desired therapeutic response.

2) Reduction in the total amount of drug administered over the period

of drug treatment.

3) Reduced blood level oscillation characteristic of multiple dosing of

conventional dosage forms.

4) Reduction in the incidence and severity of both local and systemic side

effects related to high peak plasma drug concentration.

5) Protection from first pass metabolism and gastro intestinal tract

degradation.

6) Maximizing availability with minimum dose.

7) Safety margin of high potency drugs can be increased.

8) Targeting the drug molecule towards the tissue or organ reduces the

toxicity to the normal tissues.

9) Improved patient compliance.

Increased efficacy of the drug.

Site specific delivery.

Decreased toxicity / side effects.

Increased convenience.

Shorter hospitalization.

Viable treatments for previously incurable diseases.

Potential for prophylactic application.

Lower health care costs- both short and long term.

Better patient compliance.



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1.3 DRUG DELIVERY CARRIERS (Costas Kaparissides et al., 2006)

Colloidal drug carrier systems such as micellar solutions, vesicle and

liquid crystal dispersions, as well as nanoparticle dispersions consisting of small

particles of 10–400 nm diameter show great promise as drug delivery systems

(Fig 1). When developing these formulations, the goal is to obtain systems with

optimized drug loading and release properties, long shelf-life and low toxicity. The

incorporated drug participates in the microstructure of the system, and may even

influence it due to molecular interactions, especially if the drug possesses

amphiphilic and/or mesogenic properties

Micelles formed by self-assembly of amphiphilic block copolymers (5-50

nm) in aqueous solutions are of great interest for drug delivery applications. The

drugs can be physically entrapped in the core of block copolymer micelles and

transported at concentrations that can exceed their intrinsic water- solubility.

Liposomes are a form of vesicles that consist either of many, few or just

one phospholipid bilayers. The polar character of the liposomal core enables

polar drug molecules to be encapsulated. Amphiphilic and lipophilic molecules

are solubilized within the phospholipid bilayer according to their affinity towards

the phospholipids. Participation of nonionic surfactants instead of phospholipids

in the bilayer formation results in niosomes.

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Fig 1: Pharmaceutical carriers



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Dendrimers are nanometer-sized, highly branched and monodisperse

macromolecules with symmetrical architecture. They consist of a central core,

branching units and terminal functional groups.

1.4 TARGETED DRUG DELIVERY SYSTEM (TDDS)

Drug targeting is a phenomenon (Robinson et al., 1987) in which the

distribution of drug in the body in such a manner that the major fraction of the drug

interacts exclusively with the target tissue at a cellular or subcellular level. The

objective of drug targeting is to achieve a desired pharmacological response at a

selected site without undesirable interactions at other sites.

This is especially important in cancer chemotherapy and enzyme

replacement treatment. Drug targeting is the delivery of drugs to receptors or

organs or any other specific part of the body to which one wishes to deliver the

drug exclusively.

The targeted or site specific delivery of drugs is indeed a very attractive

goal because this provides one of the most potential ways to improve the

therapeutic index of the drugs.

Earlier work done between late 1960s and the mid 1980s stressed the

need for drug carrier systems primarily to alter the pharmacokinetics of the already

proven drugs whose efficacy might be improved by altering the rates for

metabolism in liver or clearance by the kidneys. These approaches generally

were not focussed to achieve site specific or targeted delivery such as getting a

cytotoxic drug to cancerous tissue while sparing other normal, though equally

sensitive tissue. With the advancement in the carrier technology the issue of

delivering either individual drug molecule or the entire carrier to the desired site

has been addressed during the last few years.

A number of technological advances have since been made in the area of

parenteral drug delivery leading to the development of sophisticated systems that

allow drug targeting and the sustained or controlled release of parenteral

medicines.



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At present, drug targeting is achieved by one or two approaches

(Gregoriadis, 1977). The first approach involves chemical modification of the

parent compound to a derivative which is activated only at the target site.

The second approach utilizes carriers such as liposomes, niosomes,

microspheres, nanoparticles, antibodies, cellular carriers (erythrocytes and

lymphocytes) and macromolecules to direct the drug to its site of action.

Recent advancements have led to the development of several novel drug

delivery systems that could revolutionize the method of medication and provides

a number of therapeutic benefits.

The goal of any drug delivery system is to provide a therapeutic amount of

drug to the proper site in the body to achieve promptly, and then maintain, the

desired drug constant. The ideal drug delivery system delivers drug at a rate

dictated by the need of the body over the period of treatment and it channels the

active entity solely to the site of action. At present no available drug delivery

systems can achieve all these goals. The targeted drug delivery system achieves

the site specific delivery but is unable to control the release kinetics of drug in

predictable manner.

Paul Ehrlich in 1906, initiated the era of development for targeted delivery

when he envisaged a drug delivery mechanism that would target drugs directly to

diseased cells.

Number of carriers (Gregoriadis et al., 1982) were utilised to carry drug at

the target organ / tissue which include immunoglobulins, serum proteins, synthetic

polymers, lipid vesicles (liposomes), microspheres, erythrocytes, reverse

micelles, niosomes, pharmacosomes etc. Amongst the various carriers, few drug

carriers reached the stage of clinical trials where phospholipid vesicle shows

strong potential for effective drug delivery to the site of action. These carriers

(niosomes) are biologically inert in nature, devoid of any antigenic, pyrogenic or

allergic reactions and their components can be utilised as the component of



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biological membrane. Drugs incorporated in liposomes, niosomes are not

activated under physiological conditions and do not cause unfavourable side

effects as well.

There are various techniques by which drug can be targeted include

(Brahmankar et al., 2001).

1. Nanoparticles.

2. Niosomes.

3. Resealed erythrocytes.

4. Microspheres.

5. Monoclonal antibodies.

1.5 NIOSOMES AS NANOCARRIER SYSTEMS

Colloidal drug delivery systems such as liposomes and niosomes have

distinct advantages over conventional dosage forms. These systems can act as

drug reservoirs and provide controlled release of the active substance. In

addition, modification of their composition or surface can allow targeting.

Niosomes are non-ionic surfactant based vesicles that had been developed as

alternative controlled drug delivery systems to liposomes in order to overcome

the problems associated with sterilization, large-scale production and stability.

The first niosome formulations were developed and patented by L’Oreal in 1975.

They are liposome-like vesicles formed from the hydrated mixtures of cholesterol,

charge inducing substance, and nonionic surfactants such as monoalkyl or dialkyl

polyoxyethylene ether. Basically, these vesicles do not form spontaneously.

Thermodynamically stable vesicles form only in the presence of proper mixtures

of surfactants and charge inducing agents. The mechanism of vesicle formation

upon use of nonionic surfactants is not completely clear. The most common

theory is that nonionic surfactants form a closed bilayer in aqueous media based

on their amphiphilic nature. Formation of this structure involves some input of

energy, for instance by means of physical agitation (e.g. using the hand-shaking

method; Baillie et al., 1985) or heat (e.g. using the heating method; Mozafari,



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2005a Fig 2). In this closed bilayer structure, hydrophobic parts of the molecule

are oriented away from the aqueous solvent whereas the hydrophilic head comes

in contact with the aqueous solvent (Fig 3). It resembles phospholipid vesicles in

liposomes and hence, enables entrapment of hydrophilic drugs. The low cost,

stability and resultant ease of storage of nonionic surfactants has led to the

exploitation of these compounds as alternatives to phospholipids. The

superiorities and advantages of niosomes, compared to other micro and

nanoencapsulation technologies can be summarized as follows:

Compared to phospholipid molecules used in liposome formulations, the

surfactants used in the formation of niosomes are more stable

Simple methods are required for manufacturing and large–scale

production of niosomes

As the excipients and equipments used for production are not expensive,

niosome manufacturing process is cost-effective;

Niosomes possess longer shelf-life than liposomes and most other

nanocarrier systems

Unlike liposomes, they are stable at room temperature and less

susceptible to light.

Fig 2: Vesicle Formation



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Fig 3: Vesicle structure

1. Niosomes are economically cheap among the colloidal drug carrier.

2. Niosomes entrap solute in manner analogous to liposomes

(Jain et al., 1996).

3. They are more stable than liposomes, because the phospholipid, present

in the liposomes gets easily oxidized.

4. Niosomes are osmotically active and stable as well as they increase the

stability of entrapped drug.

5. They improve oral bioavailability of poorly absorbed drugs.(Biju et al., 2006)

6. They can enhance the skin penetration of drugs. (Satturwar et al., 2002).

7. They are biodegradable, biocompatible and non-immunogenic.

(Jain et al., 1994)

8. They can be made to reach the site of action by oral, parenteral as well as

topical routes.

9. Niosomes can accommodate drug molecule with a wide range of solubilities

because of the presence of hydrophilic and hydrophobic moieties together

in their structure (Jain et al., 1996).



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10. The vesicle suspension is water–based vehicle. This offers high patient

compliance in comparison with oily dosage forms.

11. The characteristics of the vesicle formulation are variable and controllable.

Altering vesicle composition, size, lamellarity, tapped volume, surface

charge and concentration can control the vesicle characteristics.

12 The vesicles may act as a depot, releasing the drug in a controlled

manner.

13 They improve the therapeutic performance of the drug molecules by

delayed clearance from the circulation, protecting the drug from biological

environment and restricting effects to target cells.

14 Niosomal dispersion in an aqueous phase can be emulsified in a non-

aqueous phase to regulate the delivery rate of drug and administer normal

vesicle in external non-aqueous phase.

1.6 TYPES OF NIOSOMES

The niosomes have been classified as a function of the number of bilayer

or as a function of size. The various types of niosomes are follows.

A) According to the nature of lamellarity (Biju. et al., 2006)

* Multilamellar vesicles (MLV) 1-5 m in size.

* Large unilamellar vesicles (LUV) 0.1 – 1μm in size

* Small unilamellar vesicles (SUV) 25 – 500 nm in size.

B) According to the size (Jain. et al., 2005)

* Small niosomes (100 nm – 200 nm)

* Large niosomes (800 nm – 900 nm)

* Big niosomes (2 μm – 4 μm)



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1.7 FACTORS AFFECTING THE FORMATION OF NIOSOMES

Type of Surfactants

Type of the surfactants influences encapsulation efficiency, toxicity, and

stability of niosomes. Initially, niosomes were formulated using cholesterol and

single-chain surfactants such as alkyl oxyethylenes. The alkyl group chain length

is usually from C12–C18. The hydrophilic- lipophilic balance (HLB) is a good

indicator of the vesicle forming ability of any surfactant Uchegbu et al., (1995,

1998) reported that the sorbitan monostearate (Span) surfactants with HLB

values between 4 and 8 were found to be compatible with vesicle formation.

Polyglycerol monoalkyl ethers and polyoxylate analogues are the most widely

used single-chain surfactants. However, it must be noted that they possess less

encapsulation efficiency in the presence of cholesterol. Etheric surfactants have

also been used to form niosomes. These types of surfactants are composed of

single-chain, monoalkyl or dialkyl chain. The latest ones are similar to

phospholipids and possess higher encapsulation efficiency. Esther type

amphyphilic surfactants are also used for niosome formulation. They are

degraded by esterases, triglycerides and fatty acids. Although these types of

surfactants are less stable than ether type ones, they possess less toxicity.

Furthermore, glucosides of myristil, cethyl and stearyl alcohols form niosomes.

Surfactant/Lipid and Surfactant/Water Ratios

Other important parameters are the level of surfactant/lipid and the

surfactant/water ratio. The surfactant/lipid ratio is generally 10–30 mM (1–2.5%

w/w). If the level of surfactant/lipid is too high, increasing the surfactant/lipid level

increases the total amount of drug encapsulated. Change in the surfactant/water

ratio during the hydration process may affect the system’s microstructure and

thus, the system’s properties.

Cholesterol

Steroids are important components of cell membranes and their presence

in membranes brings about significant changes with regard to bilayer stability,

fluidity and permeability. Cholesterol, a natural steroid, is the most commonly

used membrane additive and can be incorporated to bilayers at high molar ratios.

Cholesterol by itself, however, does not form bilayer vesicles. It is usually



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included in a 1:1 molar ratio in most formulations to prevent vesicle aggregation

by the inclusion of molecules that stabilize the system against the formation of

aggregates by repulsive steric or electrostatic effects. It leads to the transition

from the gel state to liquid phase in niosome systems. As a result, niosomes

become less leaky.

Other Additives

As is the case with liposomes, charged phospholipids such as

dicethylphosphate (DCP) and stearyl amine (SA) have been used to produce

charge in niosome formulations. The former molecule provides negative charge

to vesicles whereas the later one is used in the preparation of positively charged

(cationic) niosomes.

Nature of the Drug

One of the overlooked factors is the influence of the nature of the

encapsulated drug on vesicle formation. The encapsulation of the amphipathic

drug doxorubicin has been shown to alter the electrophoretic mobility of

hexadecyl diglycerol ether (C16G2) niosomes in a pH dependent manner,

indicating that the amphipathic drug is incorporated in the vesicle membrane.

Methods of Preparation

Methods of preparation of niosomes such as hand shaking, ether injection

and sonication in which hand shaking method forms vesicles with greater

diameter (0.3-13 µ) compared to the ether injection method

(50-1000 nm).

Small sized niosomes can be produced by Reverse Phase Evaporation

(REV) method. Microfluidization method gives greater uniformity and small size

vesicles. Niosomes by trans membrane pH gradient (inside acidic) drug uptake

process showed greater entrapment efficiency and better retention of drug.

Resistance to Osmotic Stress

Addition of a hypertonic salt solution to a suspension of niosomes brings

about reduction in diameter. In hypotonic salt solution, there is initial slow release

with slight swelling of vesicles probably due to inhibition of eluting fluid from

vesicles, followed by faster release, which may be due to mechanical loosening

of vesicles structure under osmotic stress.



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1.8 PREPARATION OF NIOSOMES

Niosomes can be prepared using non-ionic surfactants. As the number of

double layers, vesicle size and its distribution, entrapment efficiency of the

aqueous phase, and permeability of vesicle membranes are influenced by the

way of preparation, these parameters should be taken into account while making

a decision on selecting the optimum methodology for formulation. Most of the

experimental methods consist of the hydration of a mixture of the surfactant/lipid

at elevated temperature followed by optional size reduction to obtain a colloidal

dispersion. Subsequently, the unentrapped drug is separated from the entrapped

drug by centrifugation, gel filtration or dialysis. Only a couple of methods could be

found in the literature on the preparation of niosomes on an industrial scale

(Novasome®, heating method). In the Novasome® method, niosomes are

prepared upon injection of the melted surfactants/lipids into a large volume of

well-agitated, heated aqueous solutions. The novel heating method and other

well-known procedures for niosome preparation are summarized below.

Ether Injection Method

This method is essentially based on slow injection of an ether solution of

niosomal ingredients into an aqueous medium at high temperature. Typically a

mixture of surfactant and cholesterol (150 μmol) is dissolved in ether (20 mL) and

injected into an aqueous phase (4 mL) using a 14-gauge needle syringe.

Temperature of the system is maintained at 60°C during the process. As a result,

niosomes in the form of large unilamellar vesicles (LUV) are formed (Baillie et al.,

1985; Vyas and Khar, 2002).

Film Method

The mixture of surfactant and cholesterol is dissolved in an organic

solvent (e.g. diethyl ether, chloroform, etc.) in a round-bottomed flask.

Subsequently, the organic solvent is removed by low pressure/vacuum at room

temperature, for example using a rotary evaporator. The resultant dry surfactant

film is hydrated by agitation at 50–60°C and multilamellar vesicles (MLV) are

formed (Baillie et al., 1985; Varshosaz et al., 2003).



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Sonication

Typically the aqueous phase is added into the mixture of surfactant and

cholesterol in a scintillation vial. Then, it is homogenized using a sonic probe. The

resultant vesicles are of small unilamellar (SUV) type niosomes (Baillie et al.,

1986). The SUV type niosomes are larger than SUV liposomes (i.e. SUV

niosomes are >100 nm in diameter while SUV liposomes are <100 nm in

diameter). It is possible to obtain SUV niosomes by sonication of MLV type

vesicles, obtained for example through the film method explained above. For

small volume samples probe type sonicator is used while for larger volume

samples bath type sonicator is more appropriate.

Method of Handjani–Vila

Equivalent amounts of synthetic non-ionic lipids are mixed with the

aqueous solution of the active substance to be encapsulated and a homogenous

lamellar film is formed by shaking. The resultant mixture is homogenized

employing ultracentrifugation and agitation at a controlled temperature (Handjani-

Vila, 1990).

Reverse Phase Evaporation

Reverse phase evaporation technique is being used to prepare different

carrier systems including archaeosomes, liposomes, nanoliposomes and

niosomes. Typically surface-active agents are dissolved in chloroform, and 0.25

volume of phosphate saline buffer (PBS) is emulsified to get water in oil (w/o)

emulsion. The mixture is then sonicated and subsequently chloroform is

evaporated under reduced pressure. The lipid or surfactant first forms a gel and

then hydrates to form niosomal vesicles (Kiwada et al., 1985a, 1985b; Vyas and

Khar 2002). Alternatively, hydrogenated or nonhydragenated egg

phosphatidylcholine is dissolved in chloroform and PBS. The mixture is sonicated

under low pressure, forming a gel. The gel is subsequently hydrated. Free drug or

other bioactives to be encapsulated (un-entrapped material) is generally removed

by dialysis or centrifugation. Protamine is added prior to centrifugation process to

achieve phase separation.



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Heating Method

This is a non-toxic, scalable and one-step method and is based on the

patented procedure of (Mozafari 2005b). Mixtures of non-ionic surfactant,

cholesterol and/or charge inducing molecules are added to an aqueous medium

(e.g. buffer, distilled H2O, etc.) in the presence of a polyol such as glycerol. The

mixture is heated while stirring (at low shear forces) until vesicles are formed

(Mozafari 2005b).

Microfluidization

Microfluidization is a recent technique used to prepare unilamellar

vesicles of defined size distribution. This method is based on submerged jet

principle in which two fluidized streams interact at ultra high velocities, in

precisely defined micro channels within the interaction chamber. The

impingement of thin liquid sheet along a common front is arranged such that the

energy supplied to the system remains within the area of niosomes formation.

The result is a greater uniformity, smaller size and better reproducibility of

niosomes formed.

Multiple Membrane Extrusion Method

Mixture of surfactant, cholesterol and dicetyl phosphate in chloroform is

made into thin film by evaporation. The film is hydrated with aqueous

drugsolution and the resultant suspension extruded through polycarbonate

membranes which are placed in series for upto 8 passages. It is a good method

for controlling niosome size.

Transmembrane pH gradient (inside acidic) Drug Uptake

Process (remote Loading)

Surfactant and cholesterol are dissolved in chloroform. The solvent is then

evaporated under reduced pressure to get a thin film on the wall of the round

bottom flask. The film is hydrated with 300 mM citric acid (pH 4.0) by vortex

mixing. The multilamellar vesicles are frozen and thawed 3 times and later

sonicated. To this niosomal suspension, aqueous solution containing 10 mg/ml of

drug is added and vortexed. The pH of the sample is then raised to 7.0-7.2 with

1M disodium phosphate. This mixture is later heated at 60°C for 10 minutes to

give niosomes.



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The “Bubble” Method

It is novel technique for the one step preparation of liposomes and

niosomes without the use of organic solvents. The bubbling unit consists of

round-bottomed flask with three necks positioned in water bath to control the

temperature. Water-cooled reflux and thermometer is positioned in the first and

second neck and nitrogen supply through the third neck. Cholesterol and

surfactant are dispersed together in this buffer (pH 7.4) at 70°C, the dispersion

mixed for 15 seconds with high shear homogenizer and immediately afterwards

“bubbled” at 70°C using nitrogen gas.

Formation of niosomes from proniosomes

Another method of producing niosomes is to coat a water-soluble carrier

such as sorbitol with surfactant. The result of the coating process is a dry

formulation. In which each water-soluble particle is covered with a thin film of dry

surfactant (Fig 4). This preparation is termed “Proniosomes”. The niosomes are

recognized by the addition of aqueous phase at T > Tm and brief agitation.

T = Temperature.

Tm = Mean phase transition temperature.

Fig 4 : Niosome from Proniosome

Post-Preparation Processes

The main post-preparation processes in the manufacture of niosomes are

downsizing and separation of unentrapped material. After preparation, size

reduction of niosomes is achieved using one of the methods given below:



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1. Probe sonication results in the production of the niosomes in the

100–140 nm size range.

2. Extrusion through filters of defined pore sizes.

3. Combination of sonication and filtration has also been used to obtain

niosomes in the 200 nm size range (e.g. doxorubicin niosomes).

4. Microfluidization yielding niosomes in sub-50 nm sizes.

5. High-pressure homogenisation also yields vesicles of below 100 nm in

diameter.

As in most cases 100% of the bioactive agent cannot be encapsulated in

the niosomal vesicles, the unentrapped bioactive agent should be separated from

the entrapped ones.

Most commonly used methods for separating unentrapped material from

niosomes are as follows:

Dialysis

Gel filtration (e.g. Sephadex G50)

Centrifugation (e.g. 7000 × g for 30 min for the niosomes prepared by

handshaking and ether injection methods)

Ultracentrifugation (150000 × g for 1.5 h)

1. 9 CHARACTERIZATION OF NIOSOMES (Jain et al., 1994)

a) Entrapment efficiency

After preparing niosomal dispersion, unentrapped drug is separated by

dialysis, centrifugation, or gel filtration as described above and the drug remained

entrapped in niosomes is determined by complete vesicle disruption using 50% n-

propanol or 0.1% Triton X-100 and analysing the resultant solution by appropriate

assay method for the drug. Where, Entrapment efficiency (EF) = (Amount

entrapped / total amount) x 100

b) Vesicle diameter

Niosomes, similar to liposomes, assume spherical shape and so their

diameter can be determined using light microscopy, photon correlation

microscopy and freeze fracture electron microscopy.



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Freeze thawing (keeping vesicles suspension at –20°C for 24 hrs and

then heating to ambient temperature) of niosomes increases the vesicle diameter,

which might be attributed to fusion of vesicles during the cycle.

c) In vitro release

A method for in vitro release rate study includes the use of dialysis tubing.

A dialysis sac is washed and soaked in distilled water. The vesicle suspension is

pipetted into the bag and sealed. The bag containing the niosomes is placed in

200 ml of buffer solution in a 250 ml beaker with constant stirring at 25°C or

37°C. At various time intervals, the buffer is analyzed for the drug content by an

appropriate assay method.

d) Osmotic Shrinkage

Osmotic shrinkage of vesicles can be determined by monitoring

reductions in vesicle diameter, initiated by addition of hypertonic salt solution to

suspension of niosomes. Niosomes prepared from pure surfactant are

osmotically more sensitive in contrast to vesicles containing cholesterol.

e) Physical stability of vesicles at different temperature

Aggregation or fusion of vesicles as a function of temperature was

determined as the changes in vesicle diameter by laser light scattering method.

The vesicles were stored in glass vials at room temperature or kept in refrigerator

(4oC) for 3 months. The changes in morphology of multilamellar vesicles (MLVs)

and also the constituent separation were assessed by an optical microscope. The

retention of entrapped drug were measured 72 hours after preparation and after

1, 2 or 3 months in same formulations (Abbas Pardakhty et al., 2006).

f) Turbidity Measurement

The niosomes were diluted with bidistilled water to give a total lipid

concentration of 0.312 mM. After rapid mixing by sonication for 5 min, the

turbidity was measured as the absorbance with an ultraviolet-visible diode array

spectrophotometer (Fang et al., 2006).



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1.10 STABILITY OF NIOSOMES

Vesicles are stabilized based upon formation of 4 different forces:

1. Van der Waals forces among surfactant molecules

2. Repulsive forces emerging from the electrostatic interactions among

charged groups of surfactant molecules

3. Entropic repulsive forces of the head groups of surfactants

4. Short-acting repulsive forces.

Electrostatic repulsive forces are formed among vesicles upon addition of

charged surfactants to the double layer, enhancing the stability of the system.

Biological stability of the niosomes prepared with alkyl glycosides was

investigated by Kiwada et al., (1985a, 1985b). They reported that niosomes were

not stable enough in plasma. This may be due to single–chain alkyl surfactants.

SUVs were found to be more stable. Niosomes in the form of liquid crystal and

gel can remain stable at both room temperature and 4°C for 2 months. No

significant difference has been observed between the stability of these two types

of niosomes with respect to leakage. Even though no correlation between storage

temperature and stability has been found, it is recommended that niosomes

should be stored at 4°C. Ideally these systems should be stored dry for

reconstitution by nursing staff or by the patient and when rehydrated should

exhibit dispersion characteristics that are similar to the original dispersion.

Simulation studies conducted to investigate physical stability of these niosomes

during transportation to the end-user revealed that mechanical forces didn’t have

any influence on physical stability. It is assumed that the reason behind the

stability of niosomes may be due to the prevention of aggregation caused by

steric interactions among large polar head groups of surfactants.

The factors which affect the stability of niosomes are as following:

Type of surfactant

Nature of encapsulated drug

Storage temperature

Detergents

Use of membrane spanning lipids

The interfacial polymerization of surfactant monomers in situ

Inclusion of a charged molecule



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1.11 TOXICITY OF NIOSOMES

Unfortunately, there is not enough research conducted to investigate

toxicity of niosomes. Researchers measured proliferation of keratinocytes in one

of the topical niosome formulations (Hofland et al., 1991). The effect of surfactant

type on toxicity was investigated. It was determined that the ester type

surfactants are less toxic than ether type surfactants (Hofland et al., 1991, 1992).

This may be due to enzymatic degradation of ester bounds. In general, the

physical form of niosomes did not influence their toxicity as evident in a study

comparing the formulations prepared in the form of liquid crystals and gels.

However, nasal applications of these formulations caused toxicity in the case of

liquid crystal type niosomes. In some instances, encapsulation of the drug by

niosomes reduces the toxicity as demonstrated in the study on

preparation of niosomes containing vincristine (Parthasarathi et al., 1994). It

decreased the neurological toxicity, diarrhoea and alopecia following the

intravenous administration of vincristine and increased vincristine

anti-tumor activity in S-180 sarcoma and Erlich ascites mouse models.

1.12 NIOSOME DELIVERY APPLICATIONS

A variety of non-ionic surfactant vesicles have been employed as drug,

vaccine and imaging agent delivery systems. The most popular surfactants used

are the sorbitan amphiphiles (Span 20, Span 40, Span 60 and Span 80) which

incidentally are approved excipients (Rowe et al., 2003). Details follow on how

researchers have sought to exploit the ability of niosomes to control the

distribution of drug within the body for drug delivery, vaccine delivery and

diagnostic imaging.

Drug Targeting

Anti cancer drugs

Anti cancer drugs such as the model drug doxorubicin, when

encapsulated in sorbitan monostearate poly (oxyethylene) coated (coated with

Solulan C24) niosomes, circulate for prolonged periods

(Uchegbu et al., 1995). The area under the plasma level time curve is increased

six-fold by the niosomes when compared with the drug in solution, tumor levels



21

are increased by 50% and tumoricidal activity is doubled (Uchegbu et al., 1995).

These particles circulate for prolonged periods due to the poly (oxyethylene)

coating, which prevents particle recognition (Blume et al., 1993) and the uptake

by the liver and spleen (Unezaki et al., 1995). However, while a poly

(oxyethylene) coat may improve the delivery of drugs to tumors by achieving long

blood circulation times, niosomes devoid of poly (oxyethylene) coatings are also

able to improve the tumoricidal activity of drugs such as doxorubicin,

methotrexate (Chandraprakash et al., 1993) and vincristine (Parthasarathi et al.,

1994) principally by altering drug biodistribution following intravenous

administration such that the drug is targeted to some extent to the tumor tissue.

The disparate nature of the tumor vasculature (Hashizume et al., 2000) is

responsible for trapping particulate matter within tumors.

Anti infectives

The targeting of anti-leishmanial drugs to the liver, the site of pathology, is

achievable with niosomal formulations.(Baillie et al., 1985) Hexadecyl triglycerol

sodium stibogluconate niosomes are rapidly taken up by the liver producing peak

levels of antimony that are twice that achieved with the drug in solution (Baillie et

al., 1986) .The niosomes are thought to be taken up by macrophages in the liver.

The anti-parasitic activity of sodium stibogluconate is increased 10-fold by

encapsulation into niosomes (Baillie et al., 1986). However, it must be noted that

splenic and bone marrow parasites are more difficult to reach and eradicate

(Carter et al., 1988).

Delivery to the brain

Delivery of peptides to areas beyond the blood brain barrier is a major

challenge, however, there is evidence that glucose coated niosomes may be able

to achieve brain delivery of hydrophilic peptides (Dufes et al., 2004). These

vesicles are believed to exploit the glucose transporter at the blood brain barrier,

possibly by initially concentrating drug at the barrier, and have been shown to

deliver intact vasoactive intestinal peptide to the posterior and anterior parts of

the brain.



22

Topical use of niosomes

Transdermal

The topical application of niosome encapsulated drugs results in the

enhanced delivery of drugs through the stratum corneum and delivery is

specifically enhanced when hydrophilic surfactants such as poly(oxyethylene)-7-

dodecyl ether (VanHal et al., 1996) or poly (oxyethylene)-8-lauryl ester

(Honeywell-Nguyen PL et al., 2005) are used to produce flexible or "elastic"

vesicles, which have similar flexible bilayer properties to the phospholipid

transfersomes.

Ocular

Niosomal formulations for the topical treatment of glaucoma have

emerged in the form of Carbopol 934P coated sorbitan monostearate

acetazolamide niosomes (Aggarwal et al., 2004) both chitosan and Carbopol

934P coated sorbitan monostearate timolol maleate niosomes

(Aggarwal et al., 2005) and sorbitan monopalmitate timolol maleate discomes

(Vyas et al., 1998). Discome formulations with a particle size of 16/u.m produce a

sustained lowering of intraocular pressure when compared with normal niosomes

(Vyas et al., 2002). The large particle size of the discomes is believed to limit

ocular clearance. Polymer coatings are used to promote bioadhesion and to

prolong the drug residence time within the eye and the result is a

prolonged lowering of intraocular pressure. (Aggarwal et al., 2004). Chitosan is a

superior bioadhesive than Carbopol 934 in these ocular formulations

(Aggarwal et al., 2005). The acetazolamide formulations showed comparable

activity to a marketed formulation — dorzolamide (Dorzox) and activity was

sustained for up to 6 hrs (Aggarwal et al., 2004). The timolol maleate sorbitan

monostearate niosome formulation on the other hand was twice as active as a

marketed gel formulation (Aggarwal et al., 2005). All these topical niosome

formulations are able to better localize drug activity to the eye, when compared

with the drug in solution, thus minimizing deleterious systemic effects

(Aggarwal et al., 2004), (Vyas et al., 1998).



23

Niosomal vaccines

The niosomal encapsulation of both antigens (Brewer et al., 1992) and

DNA encoding for antigens results in an enhancement of the humoral (Perriea et

al., 2004) and cellular immune response to the said antigens (Brewer et al.,

1993). Although surfactants have immunostimulatory properties (Hassan et al.,

1996) the adjutancy is attributed to the actual encapsulation of the antigen

(Brewer et al., 1996) and its presentation as a particle. Enhanced protection

against an infectious challenge has also been demonstrated in mice that are

vaccinated against herpes simplex virus type 1 (Hassan et al., 1996).

Vesicles Prepared from Synthetic Amphiphiles

Niosomes as imaging agents

Targeting tumor glucose receptors is a viable method of imaging tumors.

N-palmitoyl glucosamine niosomes coated with poly (oxyethylene) and

encapsulating gadolinium salts target PC3 tumor cells on tail vein injection

(Luciani et al., 2004). Delivery to tumors is sustained as targeting was still

observed 24 hrs after dosing with the niosomes bearing both glucose and

poly(oxyethylene) units on their surface (Luciani et al., 2004). Tumor levels were

higher with the use of glycosylated, poly (oxyethylene) coated niosomes when

compared with plain sorbitan monostearate niosomes, and it is thus concluded

that the presence of either glucose or poly (oxyethylene) on the noisome surface

enables tumor targeting of the contrast agent.

Other Applications

Sustained Release

The role of liver as a depot for methotrexate after niosomes are taken up

by the liver cells. Sustained release action of niosomes can be applied to drugs

with low therapeutic index and low water solubility since those could be

maintained in the circulation via niosomal encapsulation.



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Localized Drug Action

Drug delivery through niosomes is one of the approaches to achieve

localized drug action, since their size and low penetrability through epithelium

and connective tissue keeps the drug localized at the site of administration.

Localized drug action results in enhancement of efficacy and potency of

the drug and at the same time reduces its systemic toxic effects e.g. Antimonials

encapsulated within niosomes are taken up by mononuclear cells resulting in

localization of drug, increase in potency and hence decrease both in dose and

toxicity.

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