PRESENTED BY SATYANARAYANA. V M.PHARM II-SEMISTER DEPARTMENT OF PHARMACEUTICS UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES KAKATIYA UNIVERSITY WARANGAL MULTIPLE EMULSIONS

CONTENTS  INTRODUCTION  TYPES OF MULTIPLE EMULSIONS  PREPARATION OF MULTIPLE EMULSIONS  IN VITRO CHARACTERIZATION  STABILITY OF MULTIPLE EMULSIONS  APPLICATIONS  CONCLUSION  REFERENCES

INTRODUCTION Emulsions may be described as heterogenous systems, where one immiscible liquid is dispersed in another in the form of droplets and stabilized by a third component called emulsifying agent The emulsions can be divided up into two Types : 1. Oil in water (O/W) emulsions and 2. Water in Oil (W/O) emulsions. 1. O/W Emulsion: By dispersion of oil into water the oil drops are the inner, dispersed phase. Water is the outer, continuous phase. – Aq phase – Oil drop

2. W/O Emulsion: By dispersion of water into oil the water drops are the Inner, dispersed phase. Oil is the outer, continuous phase. Oil phase Aq phase

 Multiple emulsions are complex systems in which the dispersed phase contain smaller droplets inside.  They are a type of polydispersed systems where both oil-in-water & water-in-oil emulsions exist simultaneously.  This is made possible by double emulsification hence the systems are also called as “Double emulsions”.  These are also called as “Liquid membrane systems” as the liquid film which separates the liquid phases acts as a thin semipermeable film through which solute must diffuse in order to transverse from one phase to another. MULTIPLE EMULSIONS

 Multiple emulsions are thermodynamically unstable.  They are often stabilized by using a combination of hydrophilic & hydrophobic surfactants.  The ratio of these surfactants is important in achieving stable multiple emulsions.

CLASSIFICATION  With optical microscopy method, multiple emulsions are classified as,  1. coarse (>3 micrometer in diameter)  2. Fine (1-3 micrometer in diameter)  3. Micro-multiple emulsions (<1 micrometer in diameter)

1. Water/Oil/Water (w/o/w) TYPES OF MULTIPLE EMULSIONS

1. Water/Oil/Water (w/o/w) 2. Oil/Water/Oil (o/w/o)

w/o/w

Multiple emulsions can also be formulated by using either o 1 /o 2 /o 1 systems or o 1 /o 2 /w formulations are possible. Examples of both are shown in Figure 4.

 WATER/OIL/WATER (W/O/W)  Water/Oil/Water (w/o/w) multiple emulsions consist of dispersed oil globules containing smaller aqueous droplets; each inner aqueous droplet is separated from the outer aqueous phase by an oil phase layer.  The presence of atleast two surfactants is required.  One of them is predominantly Lipophilic for stabilizing the primary w/o emulsion & the other is Hydrophilic for the secondary o/w emulsion.

 OIL/WATER/OIL(O/W/O)  Oil-in-water-in-oil (o/w/o) multiple emulsions contain an inner oil phase, a water phase, and outer oil phase.  The inner oil phase is first dispersed in water to form an oil-in-water (o/w) emulsion.  Then the o/w emulsion is further dispersed in the outer oil phase to form the o/w/o type multiple emulsion.

 Different techniques used for the preparation of Multiple Emulsions Two step emulsification technique, Phase inversion technique, Membrane Emulsification Technique,

TWO STEP EMULSIFICATION TECHNIQUE  This method involve re-emulsification of primary W/O or O/W emulsion using a suitable emulsifying agent.  Multiple emulsions, either W/O/W or O/W/O emulsions, are generally prepared using a Two-step procedure, as reported by Matsumoto et al for W/O/W emulsions, the primary emulsion (W/O) is first prepared using water and a low-HLB surfactant solution in oil.  In the second step, the primary emulsion (W/O) is re- emulsified in an aqueous solution of a high-HLB surfactant to produce a W/O/W multiple emulsion.

 The first step-that is, the preparation of the primary emulsion-is usually carried out in a high-shear device to produce a very fine droplets.  The second emulsification step is carried out in a low- shear device to avoid rupturing the multiple droplets.

 EXAMPLE  The primary W/O emulsion was prepared by adding an aqueous solution containing sodium salicylate to a Span 83 solution in light mineral oil at an equal volume ratio and stirring with a magnetic mixer (1000 rpm) for 15 minutes.  The concentration of Span 83( sorbitan sesquioleate) in light mineral oil varied from 0.1% to 40% wt/vol.  In second step, the W/O primary emulsion was re-emulsified in a Tween 80 solution containing concentrations of Tween 80 from 0.1% to 10% wt/vol at an equal volume ratio and stirred for 5 minutes at 600 rpm to produce the W/O/W multiple emulsion.

 Recently, Okochi and Nakano, reported a Modified two-step emulsification technique for the preparation of W/O/W emulsion.  This method is different from the conventional two-step technique.  Firstly, sonicated and stirring are used to produce fine, homogenous and stable W/O emulsion.  Secondly, a continuous phase is poured into a dispersed phase for preparing W/O/W emulsion.  The composition of internal aqueous phase-oily phase-external phase is fixed at 1:4:5,which produces most stable formulation of W/O/W emulsions.

PHASE INVERSION TECHNIQUE  Matsumoto and co-workers first reported the development of W/O/W system during the phase inversion of the concentrated W/O emulsion.  In this technique, an increase in volume concentration of dispersed phase may increase in phase volume ratio, which subsequently leads to the formation of multiple emulsion.  The method typically involves the addition of an aqueous phase containing the hydrophilic emulsifier examples -Tween 80 or -Sodium dodecyl sulphate or -CTAB to an oil phase consisting of liquid paraffin and containing lipophilic emulsifier ex… Span 80.

 A well-defined volume of oil phase is placed in a vessel of pin mixer.  An aqueous solution of emulsifier is then introduced to the oil phase in the vessel at a rate of 5ml/ min, while the pin mixer rotates steadily at 88 rpm at room temperature.  When volume fraction of the aqueous solution of hydrophilic emulsifier exceeds 0.7, the continuous oil phase is substituted by the aqueous phase containing a number of the vesicular globules among the simple oil droplets, leading to phase inversion and formation of W/O/W multiple emulsion.

MEMBRANE EMULSIFICATION TECHNIQUE  This method uses low shear forces to produce emulsions.  A porous glass membrane with controlled and homogenous pores is used in this method.  Particle size of the emulsion can be controlled with the proper selection of the porous glass membrane.  This is based on the use of microspores with a very narrow pore size distribution on the membrane.  The phase which is to be dispersed is pressed through membrane pores.

 The droplets formed at the membrane surface are detached by the continuous external aqueous phase flowing across the membrane surface.  To support the emulsification and prevent coalescence of droplets, a surface active compound must be added to the continuous phase  It can be successfully applied to make multiple emulsions as drug delivery systems.

Invitro characterization 1) Average globule size and size distribution, 2) Area of interfaces 3) Number of Globules 4) Creaming volume measurement 5) Conductivity test, 6) Rheological Evaluation, 7) Measurement of Zeta potential. 8) Entrapment Efficiency 9) Invitro Drug Release

 INVITRO CHARACTERIZATION  The emulsions are characterized by particle size distribution measured by -Laser diffraction -Microscopy -Image processing.  Proteins were suitable emulsifiers to create coalescence- stable multiple emulsions at low energy input.  Measuring the size distribution of the oil droplets by laser diffraction confirmed the results obtained by automated image processing.

Average globule size and size distribution  The optical microscopic method using calibrated ocular and stage micrometer can be utilized for globule size determinations of both multiple emulsions droplets as well as droplets of internal dispersed phase.  Florence and Whitchill, used inverted phase contrast microscope and a high speed camera.  The droplet size distribution of freshly made emulsion can be measured by light scattering using a - Malvern Mastersizer and - Surface mean droplet diameter and the specific surface area can be derived.

 Recently, NMR self-diffusion methods are adapted to multiple emulsion characterization.  In addition, the self diffusion NMR technique may be used to obtain information regarding water exchange across the oil film in the multiple emulsions.

Area of interfaces  The average globule diameter determined can be used in the calculation of the total surface area of interface using S = 6/D S = total area of interface (sq cm) D = diameter of globule (cm)

Number of globules  Number of globules per cubic mm can be measured by using the Haemocytometer cell No. of globules ×dilution × 4000 No. of globules/mm 3 = No. of small squares counted.

 Creaming volume measurement  The creaming volume was defined as the relative difference in volume of the multiple emulsion and the volume of the creamed phase. V multiple emulsion –V creamed phase % V cream = × 100 V multiple emulsion.

Conductivity test  This test was found to be an important parameter to study the stability and yield of W/O/W emulsions.  The conductivity was determined by means of a systronics digital conductivity meter. The entrapment percent / yield value (E%) was calculated according to the following equation E% = 100. (Ci – Ct )/ Ci Ci = conductivity of the internal aqueous phase Ct = conductivity value of multiple emulsion at a given time t.

Rheological evaluation  The rheology of multiple emulsion is an important parameter as it relates to emulsion stability and clinical performance.  The Viscosity and Interfacial elasticity are two major parameters, which relate to product rheology.  Viscosity (non-newtonian)– by Brookfield rotational viscometer.  Oil phase viscosity – by Stress viscometry using a Behlin controlled rheometer.

 Three different geometries stant(5 µl) were used according to viscosity of samples Double gap DG cone plate 4/40 concentric cylinder c25  Interfacial film strength – evaluated by interfacial film measurements by Oscillatory surface rheometer, i.e. elasticity of w/o and o/w components of w/o/w multiple emulsions and these data may relate to emulsion stability.

Measurement of zeta potential  The zeta potential and surface charge can be calculated using Smoluchowski’s equation from the mobility and electrophoretic velocity of dispersed globules using zeta potentiometer.  Nakhare and Vyas, using a cylindrically bored micro electrophoresis cell equipped with platinum-irradium electrodes to measure the electrophoretic mobility of the diluted w/o/w emulsion and using the following equation zeta potential was calculated.

ζ = 4μηП/٤E Where, ζ = zeta potential η= viscosity of the dispersion medium ٤=dielectric constant of the dispersion medium E=potential gradient ( voltage applied/distance b/w electrodes) μ=migration velocity

Entrapment efficiency Entrapment efficiency of the system is a measure of drug loading capacity of the system. It is necessary to estimate the drug inside the multiple emulsion and in the aqueous phase. Analysed weight of drug in multiple emulsion Drug entrapment efficiency (%) = ×100 Theoretical weight of drug loaded in system

In-vitro drug release The drug release from the aqueous phase of a W/O/W emulsion can be estimated using the conventional dialysis technique. Nakhare and vyas, 1995 investigated the release the dialysis method using cellophane tubing. The W/O/W emulsion was placed in the dialysis bag and dialyzed against 200 ml of phosphate saline buffer (PBS, P H 7.4) at 37±1 o C and a sink condition was maintained while sink contents were stirred continuously using a magnetic stirrer. Aliquots were withdrawn at different time intervals and estimated using standard procedure and the data were used to calculate cumulative drug release profile

STABILITY OF MULTIPLE EMULSIONS Emulsion stability is a phenomenon, which depends upon the equilibrium between water, oil and surfactant. Multiple emulsions are thermodynamically unstable. The possible indications of instability include: Leakage of the contents from the inner aqueous phase. Expulsion of internal droplets in external phase. Constriction or distension of the internal droplets due to osmotic gradient across the oil membrane. Flocculation of internal aqueous phase and multiple emulsion droplets. Disruption of oil layer on the surface of internal droplets. Phase separation.

 APLLICATONS OF MULTIPLE EMULSIONS  Multiple emulsions finds wide range of applications in controlled or sustained drug delivery, targeted delivery, taste masking, bioavailability enhancement, enzyme immobilization, etc.  it will be able to provide a novel carrier system for drugs, cosmetics and pharmaceutical agents.

1. Hemoglobin multiple emulsion having specified properties is suitable for provision of oxygen as a blood substitute and other oxygen transfer processes. 2. W/O/W multiple emulsions are systems of potential interest in the oral administration of insulin. Although it has been shown that a single oral administration of insulin-loaded W/O/W multiple emulsion to diabetic rats led to the significant decrease of blood glucose.

3. Water-in-oil-water (w/o/w) multiple emulsions and polymeric nanoparticle formulations containing influenza virus surface antigen hemagglutinin (HA) are thought to be suitable carriers for a vaccine delivery system. 4. Using a water-in-oil-in-water multiple emulsion system developed for pulmonary drug targeting, the effectiveness of tetrandrine as an antifibrotic agent

 5. To develop a prolonged and sustained release preparation, an albumin micro-sphere-in-oil-in- water emulsion was prepared. Tegafur was used as a model drug.  6. Vitamin c has been widely used in formulations of skin care products.  7. Multiple emulsions are also used in topical application ex… a w/o/w emulsion of Metronidazole.

Marketed Multiple Emulsions 1 Sandostatin LAR TM Depot – Novartis (hypothalamic hormones analogue) Control of hypersecretion at the site of the tumor where hormone overproduction starts 2. IVY FORMULA 30 Main Treatment Multiple Emulsion- IVY FORMULA 30 was created as a futuristic skin care treatment that is concentrated into four short weeks. A 4-week concentrated treatment that restores the keratinization cycle and awakens and boosts the skin’s “underlying strength.”-marketed by IVY Cosmetics

CONCLUSION Multiple emulsions known to be promising delivery systems for both pharmaceuticals and cosmetic materials. The possibility of encapsulating active substances within liquid membranes may lead to interesting opportunities in both fields. Thus the formulation, manufacturing, stabilization, analysis and potential application of multiple emulsions seems to be worth surveying, putting a special emphasis on cosmetic applications.

REFERENCES 1) Pharmaceutical Emulsions and Suspensions, edited by Francoise Nielloud Gilberte Marti-Mestres. 2) Pharmaceutical Dosage Forms: Disperse systems, vol.3, second edition, Edited by Herbert A.Liberman, Martin M. Rieger and Gilbert S. Banker. 3) some studies on Multiple Emulsions by G.Vishwanadham, (Ph.D thesis, KU) 4) Advances in Controlled and Novel Drug Delivery Systems, Edited by N.K. Jain. 5) Targeted and controlled Drug Delivery Novel Carrier Systems by -S.P. Vyas and R.K. Khar

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