NIOSOMES: A NOVEL DRUG DELIVERY SYSTEM Presented by: Anirban Saha M.Pharm (Pharmaceutics) Amity Institute of Pharmacy (AIP) AMITY INSTITUTE OF PHARMACY
Jul 16, 2015
NIOSOMES: A NOVEL DRUG
DELIVERY SYSTEM
Presented by:
Anirban Saha
M.Pharm (Pharmaceutics)
Amity Institute of Pharmacy (AIP)
AMITY INSTITUTE OF
PHARMACY
Introduction
Factors Affecting Niosomes Preparation
Methods of Preparation
Characterization of Niosomes
Stability of Niosomes
Applications of Niosomes
Toxicity of Niosomes
PRESENTATION FLOW
NOVEL DRUG DELIVERY SYSTEM (NDDS)
Refers to approaches, formulations, technologies, and
systems for transporting a pharmaceutical compound in the
body as needed to safely achieve its desired therapeutic
effect
May involve scientific site-targeting within the body, or
facilitating systemic pharmacokinetics
Technologies modify drug release profile, absorption,
distribution and elimination for the benefit of
Improving product efficacy and safety
Patient convenience and compliance
INTRODUCTION
EXAMPLES OF NDDS
• Niosomes
• Liposomes
• Nanoparticles
• Resealed erythrocytes
• Microspheres
• Monoclonal antibodies
• Micro emulsions
• Antibody-loaded drug delivery
• Magnetic microcapsules
• Implantable pumps
Figure 1: various drug delivery systems (Aitha S, 2013)
Novel drug delivery system, in
which the medication is
encapsulated in a vesicle which is
composed of a bilayer of non-ionic
surface active agents (Nasir A, 2012)
Are very small, and microscopic in
size.
Although structurally similar to
liposomes, they offer several
advantages over them.
NIOSOMES
Figure 2: Niosomes Vesicles (Aitha S, 2013)
The vesicles forming
amphiphile is a non-ionic
surfactant stabilized by
addition of cholesterol and
small amount of anionic
surfactant such as dicetyl
phosphate
NIOSOMES
Figure 3: Vesicle of niosome (Aitha S, 2013)
Figure 4: Structure of Niosomes
STRUCTURE
OF NIOSOMES similar to liposomes, in that they are also
made up of a bilayer.
However, the bilayer in the case of
Niosomes is made up of non-ionic
surface active agents rather than
phospholipids.
Made of a surfactant bilayer with its
hydrophilic ends exposed on the outside
and inside of the vesicle, while the
hydrophobic chains face each other
within the bilayer.
(Patel SM et al, 2012)
(Makeshwar KB, 2013)
STRUCTURE
OF NIOSOMES
vesicle holds hydrophilic
drugs within the space
enclosed in the vesicle,
while hydrophobic drugs
are embedded within the
bilayer itself.
Niosomes vesicle would
consist of a vesicle
forming amphiphile i.e. a
non-ionic surfactant such
as Span- 60, which is
usually stabilized by the
addition of cholesterol
(Makeshwar KB, 2013)
Figure 5: Structure of niosome (Makeshwar KB, 2013)
Entrap solutes in a manner analogous to liposomes.
Osmotically active and stable.
Accommodate the drug molecules with a wide range ofsolubility.
Exhibits flexibility in their structural characteristics(composition, fluidity and size)
Performance of the drug molecules is increased.
Better availability to the particular site by protecting thedrug from biological environment.
Surfactants used in preparation are biodegradable,biocompatible and non-immunogenic
SALIENT FEATURES OF
NIOSOMES (Makeshwar KB, 2013)
Improve the therapeutic performance of the drug molecules by
Delayed clearance from the circulation
Protecting the drug from biological environment
Restricting effects to target cells
Niosomal dispersion in an aqueous phase can be emulsified in a
nonaqueous phase to
Regulate the delivery rate of drug
Administer normal vesicle in external non-aqueous phase.
Handling and storage of surfactants requires no special conditions.
Bioavailability of poorly absorbed drugs is increased.
Targeted to the site of action by oral, parenteral as well as topical
routes.
ADVANTAGES OF NIOSOMES
DELIVERY SYSTEM (Makeshwar KB, 2013)
According to the nature of lamellarity
1. Multilamellar vesicles (MLV) 1-5 μm in size.
2. Large Unilamellar vesicles (LUV) 0.1 – 1μm in size
3. Small Unilamellar vesicles (SUV) 25 – 500 nm in size.
According to the size
1. Small Niosomes (100 nm – 200 nm)
2. Large Niosomes (800 nm – 900 nm)
3. Big Niosomes (2 μm – 4 μm)
TYPES OF NIOSOMES
FACTORS AFFECTING THE
FORMATION OF NIOSOMES
Type of surfactant influences encapsulation efficiency,
toxicity, and stability of Niosomes
NATURE OF SURFACTANT
The surfactant/lipid ratio is generally 10-30 mM (1-2.5%
w/w)
Increasing the surfactant/lipid level increases the total
amount of drug encapsulated
SURFACTANT AND LIPID LEVELS
NATURE OF THE DRUG
The Physio-chemical propertiesof encapsulated drug influencecharge and rigidity of theNiosome bilayer.
The drug interacts withsurfactant head groups anddevelops the charge that createsmutual repulsion betweensurfactant bilayers, and henceincreases vesicle size.
The aggregation of vesicles isprevented due to the chargedevelopment on bilayer.
Effect of the nature of drug on
formation vesicle
CHOLESTEROL(Tamizharas S et al, 2009)
Addition of cholesterol molecule to
Niosomal system
• Makes the membrane rigid
• Reduces leakage of drug from the Niosome
• Increases the chain order of bilayer
• Strengthen the non-polar tail of the non-ionic
surfactant
• Increase in the entrapment efficiency
• Leads to the transition from the gel state to
liquid phase in Niosomes systems
MEMBRANE ADDITIVES
Cholesterol
Charge inducers are one of the membrane
additives which are often included in Niosomes
because
Increase surface charge density
Prevent vesicles flocculation, Aggregation and
Fusion.
Examples: Dicetyl phosphate (DCP) and Stearyl
amine (SA)
MEMBRANE ADDITIVES(Nasir A, 2012)
Film Method
Ether Injection Method
Sonication
Reverse Phase Evaporation
Heating Method
Microfluidization
Multiple Membrane Extrusion Method
Transmembrane pH gradient (inside acidic) DrugUptake Process (remote Loading)
The “Bubble” Method
Formation of Niosomes from Proniosomes
METHODS OF PREPARATION (Madhav NVS, 2011)
•Mixture ofSurfactant andCholesterol
Dissolved in an organic solvent
in a round-bottomed flask.
(e.g. diethyl ether,
chloroform, etc.)
•organic solvent isremoved by lowpressure/vacuum atroom temperature
example using a rotary evaporator.
• The resultantdry surfactantfilm is hydratedby agitation at50–60°C
Multilamellar vesicles
(MLV) are formed
FILM METHOD • Also known as hand shaking method
FILM METHOD
Figure 6: Steps of Film method (Madhav NVS, 2011)
A solution of the surfactant ismade by dissolving it in diethylether.
This solution is then introduced using aninjection (14 gauge needle) into warm wateror aqueous media containing the drugmaintained at 60°C.
Vaporization of the etherleads to the formation ofsingle layered vesicles.
• The particle size of the Niosomes formed depend on theconditions used, and can range anywhere between 50-1000μm. (Madhav NVS, 2011)
ETHER INJECTION METHOD
Figure 7: Steps of Ether injection method (Madhav NVS, 2011)
The mixture is probe sonicated
at 60°C for 3 minutes using a sonicator with a titanium probe to yield Niosomes.
Added to the surfactant/ cholesterol mixture in a 10 ml glass
vial
Aliquot of drug solution in buffer
SONICATION
Figure 8: Sonication method (Madhav NVS, 2011)
Creation of a solutionof cholesterol andsurfactant (1:1 ratio)in a mixture of etherand chloroform
An aqueous phasecontaining the drugto be loaded isadded to this
Resulting twophases aresonicated at 4-5°C
A clear gel isformed which isfurther sonicatedafter the additionof phosphatebuffered saline(PBS)
Temperature israised to 40°C andpressure is reducedto remove theorganic phase
Viscous Niosomesuspension is formedwhich can be dilutedwith PBS and heatedon a water bath at60°C for 10 minutesto yield Niosomes
REVERSE PHASE EVAPORATION
Non-toxic, Scalable and one-step method.
HEATING METHOD
Mixtures of non-ionicsurfactant, cholesteroland/or charge inducingmolecules are added to anaqueous medium e.g.buffer, distilled H2O, etc
• In the presence of aPolyol such as glycerol.
The mixture is heated while stirring at low shear forces
• Until vesicles are formed
Recent technique used to prepare Unilamellar vesicles of
defined size distribution.
based on submerged jet principle
MICROFLUIDIZATION
Two fluidizedstreams interact atultra high velocities,in precisely definedmicro channelswithin the interactionchamber
The impingement of thinliquid sheet along acommon front is arrangedsuch that the energysupplied to the systemremains within the area ofNiosomes formation
The result is a greateruniformity, smallersize and betterreproducibility ofNiosome are formed
MICROFLUIDIZATION
Figure 9: Steps of microfludization method (Madhav NVS, 2011)
Good method for controlling Niosomes size.
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 drug solution
Resultant suspension is extruded through polycarbonate membranes which are placed in series for upto 8 passages
Figure 10: Multiple membrane
extrusion method (Madhav NVS, 2011)
Solution of surfactantand cholesterol is madein chloroform
Solvent is then evaporatedunder reduced pressure to geta thin film on the wall of theround bottom flask, similar tothe hand shaking method
This film is thenhydrated using citric acidsolution by vortexmixing
Resulting Multilamellar vesicles are then treated
to three freeze thaw cycles and sonicated
To the Niosomal suspension, aqueous solution containing 10mg/ml of drug is added and vortexed
pH of the sample is then raised to 7.0-7.2 using 1M disodium
phosphate
Mixture is heated at 60°C for 10 minutes to
give Niosomes
TRANSMEMBRANE pH GRADIENT DRUG
UPTAKE PROCESS
A recently developed technique which allows the preparation of
Niosomes without the use of organic solvents.
BUBBLE METHOD
The bubbling unit consists of a round bottom flask with three necks, and this is positioned in a water bath to control the temperature.
Water-cooled reflux and thermometer is positioned in the first and second neck, while the third neck is used to supply nitrogen.
Cholesterol and surfactant are dispersed together in a buffer (pH 7.4) at 70°C.
This dispersion is mixed for a period of 15 seconds with high shear homogenizer and immediately afterwards, it is bubbled at 70°C using the nitrogen gas to yield Niosomes.
FORMATION OF NIOSOMES FROM
PRONIOSOMES (Makeshwar KB, 2013)
Water soluble carrier such as
sorbitol is coated 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.
This preparation is termed
“Proniosomes”.
The Niosomesare recognized bythe addition ofaqueous phase atT > Tm and briefagitation.
T=Temperature.Tm = mean phase transition temperature
POST-PREPARATION PROCESSES
1) Dialysis:
The aqueous niosomal dispersion is dialyzed in a dialysis tubing
against phosphate buffer or normal saline or glucose solution.
2) Gel Filtration:
The unentrapped drug is removed by gel filtration of niosomal
dispersion through a Sephadex-G -50 column and elution with
phosphate buffered saline or normal saline.
3) Centrifugation:
The niosomal suspension is centrifuged and the supernatant is
separated. The pellet is washed and then resuspended to obtain a
niosomal suspension free from unentrapped drug.
POST-PREPARATION PROCESSES (Makeshwar KB, 2013)
a) Size, Shape and Morphology
b) Entrapment efficiency
c) Vesicle diameter
d) In vitro release
e) Vesicle charge
f) Bilayer rigidity and Homogeneity
g) Osmotic Shrinkage
h) Physical stability of vesicles at different temperature
i) Turbidity Measurement
CHARACTERIZATION OF NIOSOMES
Structure of surfactant based vesicles has been visualized
and established using freeze fracture microscopy
Photon correlation spectroscopy used to determine mean
diameter of the vesicles.
Electron microscopy used for morphological studies of
vesicles
Laser beam is generally used to determine size distribution,
mean surface diameter and mass distribution of Niosomes.
SIZE, SHAPE AND MORPHOLOGY
After preparing Niosomal dispersion, unentrapped drug is
separated by
Dialysis
Centrifugation
Gel filtration
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. (Bragagnia M, 2012)
ENTRAPMENT EFFICIENCY
To determine drug loading and encapsulation efficiency,
the niosomal aqueous suspension was ultracentrifuged,
supernatant was removed and sediment was washed
twice with distilled water in order to remove the
adsorbed drug.
The Niosomal recovery was calculated as:
NIOSOMAL DRUG LOADING
(Makeshwar KB, 2013)
Niosomes diameter can be determined using
Light microscopy
Photon correlation microscopy
Freeze fracture electron microscopy.
Freeze thawing
VESICLE DIAMETER (Shirsand SB, 2012)
Figure 11: Microphotograph of niosomes (Shrisand SB, 2012)
At various time intervals, the buffer is analysed for the drug content by an appropriate assay method.
The bag containing the vesicles is placed in 200 ml of buffer solution in a 250 ml beaker with constant shaking at 25°C or 37°C.
The vesicle suspension is pipetted into a bag made up of the tubing and sealed.
A dialysis sac is washed and soaked in distilled water.
A method of in-vitro release rate study includes the use of dialysis tubing.
IN VITRO RELEASE (Makeshwar KB, 2013)
The vesicle surface charge can play an important role in thebehaviour of Niosomes in vitro and in vivo.
Charged Niosomes are more stable against aggregation andfusion than uncharged vesicles.
In order to obtain an estimate of the surface potential, the zetapotential of individual Niosomes can be measured byMicroelectrophoresis, Fluorophores, and Dynamic lightscattering.
Zeta potential is calculated by using Henry equation (S P Vyas, 2011)
ζ =µ𝐸4πη
ΣWhere ζ is Zeta potential, µ𝐸 is electrophoretic mobility, η isviscosity of the medium and Σ is dielectric constant
VESICLE CHARGE
(Makeshwar KB, 2013)
The biodistribution and biodegradation of Niosomes are
influenced by rigidity of the bilayer.
Homogeneity can occur both within Niosomes structures
themselves and between Niosomes in dispersion and
could be identified via. NMR, Differential Scanning
Calorimetry (DSC) and Fourier transform-infra red
spectroscopy (FT-IR) techniques.
Membrane rigidity can be measured by means of
mobility of fluorescence probe as a function of
temperature. (Patel SM et al, 2012)
BILAYER RIGIDITY AND HOMOGENEITY
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.
OSMOTIC SHRINKAGE
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
PHYSICAL STABILITY OF VESICLES
AT DIFFERENT TEMPERATURE
Niosomes were diluted with bidistilled water to give a total
lipid concentration of 0.312 mM
After rapid mixing by sonication for 5 min
Turbidity was measured as the absorbance with an
ultraviolet-visible diode array spectrophotometer.
TURBIDITY MEASUREMENT
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.
STABILITY OF NIOSOMES
FACTORS
Nature of surfactant
Structure of surfactant
Temperature of hydration
Nature of encapsulate
d drug
Inclusion of a charged molecule
FACTORS AFFECTING STABILITY OF NIOSOMES
A surfactant used for preparation of Niosomes must have ahydrophilic head and hydrophobic tail.
The hydrophobic tail may consist of one or two alkyl orperfluoroalkyl groups or in some cases a single steroidalgroup.
The ether type surfactants with single chain alkyl ashydrophobic tail is more toxic than corresponding dialkyletherchain.
The ester type surfactants are chemically less stable than ethertype surfactants and the former is less toxic than the latter dueto ester-linked surfactant degraded by esterases totriglycerides and fatty acid in vivo.
The surfactants with alkyl chain length from C12-C18 aresuitable for preparation of Niosome.
NATURE OF SURFACTANT (Singh CH, 2011)
The geometry of vesicle to be formed from surfactants is affected by itsstructure, which is related to critical packing parameters
Critical packing parameters can be defined using following equation,
𝐶𝑝𝑝 =𝑣
lc∗ a0
Where
v = hydrophobic group volume,
lc = the critical hydrophobic group length
a0 = the area of hydrophilic head group
From the critical packing parameter value type of miceller structureformed can be ascertained as given below,
If CPP < ½ then formation of spherical micelles,
If ½ < CPP < 1 formation of bilayer micelles,
If CPP > 1 formation inverted micelles23.
surfactants with longer alkyl chains generally give larger vesicles
STRUCTURE OF SURFACTANT
(Madhav NVS, 2011)
The physico-chemical properties of encapsulated drug
influence charge and rigidity of the Niosome bilayer.
The drug interacts with surfactant head groups and
develops the charge that creates mutual repulsion between
surfactant bilayers and hence increases vesicle size.
NATURE OF ENCAPSULATED DRUG(Singh CH, 2011)
Hydration temperature influences the shape and size of
the Niosome.
For ideal condition it should be above the gel to liquid
phase transition temperature of system.
Temperature change of Niosomal system affects
assembly of surfactants into vesicles and also induces
vesicle shape transformation
TEMPERATURE OF HYDRATION (Madhav NVS, 2011)
Niosomes as Drug Carriers
Diagnostic imaging with Niosomes
Drug Targeting
Delivery to the brain
Anti cancer drugs
Anti infectives
Targeting of bioactive agents
To Reticulo-endothelial system (RES)
To organs other than RES
NIOSOME DELIVERY APPLICATIONS(Malhotra M et al, 1994)
Ophthalmic drug delivery
Delivery of peptide drugs
Immunological application of Niosomes
Transdermal delivery of drugs by Niosomes
Delivery system for the vasoactive intestinal peptide
(VIP)
Niosomes as carriers for Hemoglobin
Niosomal vaccines
NIOSOME DELIVERY APPLICATIONS
Sustained Release
Localized Drug Action
OTHER APPLICATIONS
Unfortunately, there is not enough research conducted to
investigate toxicity of Niosomes.
It was determined that the ester type surfactants are less
toxic than ether type surfactants.
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.
Nasal applications of these formulations caused toxicity in
the case of liquid crystal type Niosomes.
TOXICITY OF NIOSOMES
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REFERENCE