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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.
7
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
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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.
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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).
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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.