route of administration of biotech products

Pharmaceutical Biotechnology Fall 2015

Introduction:

The field of biotechnology does not deal with a specific mode of drug delivery, rather a

category of products that are defined by their source and methods of production.

Pharmaceutical biotechnology uses living organisms or biological processes to

manufacture or modify pharmaceutical products. The chemical properties of many

biotechnology-derived compounds dictate the route of administration which is typically

parenteral. Other non-oral routes are also becoming increasingly important. The oral route

is generally avoided because many biotechnology products are destroyed by gastric acid,

rapid metabolism via the liver, or elimination via the kidneys (Desai and Lee, 2007).

In recent years, the number of drugs of biotechnological origin available for many

different diseases has increased exponentially, including different types of cancer,

diabetes mellitus, infectious diseases (e.g. AIDS Virus / HIV) as well as cardiovascular,

neurological, respiratory, and autoimmune diseases, among others. The pharmaceutical

industry has used different technologies to obtain new and promising active ingredients,

as exemplified by the fermentation technique, recombinant DNA technique and the

hybridoma technique. The expiry of the patents of the first drugs of biotechnological

origin and the consequent emergence of biosimilar products, have posed various

questions to health authorities worldwide regarding the definition, framework, and

requirements for authorization to market such products (Almeida et al., 2011).

In recent years, the focus of pharmaceutical research is gradually shifting to the

development of drug delivery systems rather than finding newer chemical entities for an

all-round improvement in drug therapy. Multiferous materials and principles have been

employed to generate a wide variety of carrier classes such as polymer-based particulate

systems (microspheres, microcapsules, nanoparticles, and transdermal patches) and rigid,

semi-rigid and vescicular lipoidal colloid drug delivery vehicles (liposomes, niosomes,

microemulsions, micelles).In the above approaches, the latter has achieved a favorable

position because of their non particulate nature, components of bio-origin and provisions

for modifications in the constructs so that they can be tailored to suit the target site

(Surabhi et al., 2010).

Route of Administration of Biotech Products: Parenteral Route Considering Nano-particle & Microemulsion


Biotech Products or Biotechnology Derived Products:

Biotech is the term used for biotechnology or products produced by biotechnology. These

drugs are also called biological, biotech drugs, biological drugs, or biopharmaceuticals.

True biotech products are manufactured in live biological systems known as expression

systems. Biopharmaceuticals are physically very different from small molecule drugs

generally sized at 1 kDa. Biotechnology products in medicine are manufactured by using

recombinant DNA technology, which entails genetic manipulation of cells, or a

monoclonal antibody. Biopharmaceutical forms are potent, reactive, unstable and very

expensive (Bruggemeier, 2006; Rader, 2008). They have several advantages such as the

provision of effective treatments in chronic and uncommon diseases. Recombinant drugs

(Factor VIII for hemophilia), offer safer and reduced side effects, improve on existing

therapies and can be produced on a large scale by biotechnological processes (Almeida et

al., 2011).

Examples of drugs obtained by biotechnology processes:

Antibiotic

Blood factors

Hormones

Cytokines

Growth factors

Enzymes

Monoclonal antibodies

Vaccines

Routes of Administration of Biotech Products:

The route of administration of a biotech drug is very different from a traditional drug

taken as a pill or capsule, and each drug is developed with a unique route of

administration. These drugs are mainly given intravenously, subcutaneously or

intramuscularly. There are also biotech drugs given to patients by intrathecal,



intraarticular, and inhalation routes. They cannot be given orally because they would be

degraded in the gastrointestinal tract. Most biotech drugs are given in the clinic but for

some chronic indications the trend is to develop subcutaneous versions so that they can be

self-administered at home with an auto injector device (e.g. Insulin).

Routes of administration of biotech products are:

Oral route.

Parenteral route.

Nasal route.

Transmucosal route.

Impact of Route of Administration on Therapeutic Outcome of Biotech Products:

The advantage of oral drug delivery is very clear from the standpoint of both

manufacturing and patient convenience. The chemical structure of the biotechnological

derived drugs developed to date that means they are extremely susceptible to degradation

to gastrointestinal tract. In addition because of their large size and poor membrane

permeation, they cannot easily cross the walls of gastrointestinal tract in sufficient

amounts to be useful.6 On the other hand, vaccines still proteins, may be given orally,

because only small amount of the dose given is needed to elicit and immune response and

the mucosal immune response associated with oral delivery of vaccine is useful in

preventing disease of fecal oral transmission (e.g. polio).Methods of improving the

bioavailability of peptides drugs are currently being investigated. This mainly involves

the inclusion of absorption enhancers and enzyme inhibitors in the drug formulation. So

far these methods have met little success. The same properties that prohibit oral

administration of proteins also limit their delivery via several other routes of

administration. However, the use of absorption enhancers may increase their

bioavailability when given by non-injection routes (nasal, buccal, rectal, transdermal

6).Pulmonary delivery of protein does not have the same bioavailability limitations as

other routes and several proteins formulated for delivery via this route undergoing clinical

trials. An inhaled form of insulin Exubera was approved in January, 2006. Degradation is

less than with oral delivery because the lungs have less proteolytic activity less than the

gastrointestinal tract and pulmonary administration bypasses first-pass hepatic



metabolism of drugs. In addition, larger molecules such as insulin cross the aveoli via

passive diffusion better than they cross the other membranes. Special inhalation devices

must be used for optimal delivery. The limited usefulness of most routes of administration

has meant that virtually all currently marketed biotech products are given via the

parenteral routes. The large size of proteins is not a factor with this method of delivery

and although enzymes may still be present in bloodstream to degrade the drugs, sufficient

quantities may be delivered to achieve desired effects (Desai and Lee, 2007).

Parenteral Administration of Biotechnology Derived Drugs:

The vast majority of biotechnological derived drugs are delivered to the patient by

injection. Proteins are chemically unstable and giving them orally may be lead to

degradation in gastrointestinal tract. They are large molecules with poor membrane

permeability characteristics, factors that make non-injection methods more difficult.

Although injectable formulations make delivery of biotechnology derived drugs possible,

other issues must be addressed.

Figure 1: Parenteral Route of Administration

The half life of proteins can be extremely short, so that the drug has insufficient time to

circulate in the body. Several approaches have been taken to address the residence time



problem, including creating a depot effect by giving the drug intramuscular or

subcutaneous routes instead of the intravenous routes (e.g. leuprolide). The other

significant approach is chemical modification, either modification of the protein itself

using either site directed mutagenesis to form chemically modified proteins with longer

half lives or addition or modification of side chains by glycolysation or pegylation of the

protein. Intravenous administration allows the highest concentration of the drug to be

achieved, because any pre-systemic degradation is avoided. This is not always a

convenient method of drug delivery, however and it is best use with drugs in an inpatient

setting that need to reach peak blood levels quickly. The tissue plasminogen activators are

good examples. Subcutaneous administration is common with biotechnology derived

drugs, particularly those used to treat chronic conditions, because the patients can be

taught to self-administer the product. Three rheumatoid arthritis products (etanercept,

anakinra and adalimumab) are available as prefilled syringes for subcutaneous injections.

The molecular weight plays a major role in determining where subcutaneously

administered drugs are absorbed. Drugs can be given less frequently than when given

intravenously and in some cases the intramuscular routes may be preferred to the

subcutaneous route.

Advantages of Parenteral Routes of Administration:

Rapid action of drug.

Can be employed in unconscious/ uncooperative patients.

Drugs, which are not absorbed in small intestine or irritate the stomach can be

administered by this route.

Drugs, which are modified by alimentary juices and liver can be given by this route.

Can deliver drugs in large amounts, will have 100 percent bioavailability.

Does not have 1st pass metabolism.

Polar drug can be given as they are absorbed (e.g. - Streptomycin).

Gastric irritation and vomiting are provoked.



Disadvantages of Parenteral Routes of Administration:

It is generally more risky.

The preparation must be sterile.

The technique is intensive and painful.

Drug administered by all routes except intra-arterial might still.

Eliminated by first pass elimination in liver prior to distribution to the rest of the

body.

Particulate Carrier Systems Used for Drug Products by Parenteral Route:

In search of safe and effective therapy, the development of new drugs has been the

common practice historically. However, it involved a long gestation period in terms of

time, efforts, and huge cost. Later on, it was realized that the issues pertaining to efficacy

and safety are largely influenced by the distribution of the drug within the biological

system, as there is appreciable deviation from the desired site of action, i.e., the target

site. This objective, hitherto un-accomplished gave way to an alternate approach of drug

delivery, wherein the carrier systems were used to deliver the molecules to specific

receptor sites without afflicting the normal tissues and organs of the body. The

fundamentals lie in hosting the drug in carefully designed carriers to bring favorable

change(s) in its surrounding microenvironment, and consequently, its delivery. It is the

modification(s) in physicochemical characteristics of the molecules and in the barrier

properties of the biological membranes at various locations, which lead to improved

transportation of drugs toward the diseased locations. Further, it improves the chances of

the availability of the drug at the specific receptor site and enhances drug receptor

interaction through mediation of specialized composition and design of the carrier

systems. All these factors tend to potentiate the degree of pharmacodynamic response, the

safety and patient compliance being the immediate release (Jha et al., 2011). There are

different types of particulate carrier systems .These are:

Microspheres

Microcapsules



Nanoparticles

Aquasomes

Liposomes

Micromulsions

Cellular carriers

Replication defective viruses.

Microemulsion:

Micro-emulsion is homogeneous, transparent, thermodynamically stable dispersions of water

and oil, stabilized by a surfactant, usually in combination with a co-surfactant. In this type of

system, the two liquids tend to separate out in two layers, and to avoid this, a third substance

called as an emulsifier is added which is, in general, surface-active agent or surfactant.

Surfactant molecules contain both a polar and a non polar group. So they exhibit a very

peculiar behavior, first, they tend to adsorb at interface, where they can fulfill their dual

affinity with hydrophilic groups located in aqueous phase and hydrophobic groups in oil or

air. Second, they reduce the mismatch with solvent through a specific kind of aggregation

process known as micellization (Giri et al., 2013).



Figure 2: Composition of Microemulsion

Background of Microemulsion:

Microemulsions were not really recognized until the work of Hoar and Schulman in 1943,

who reported a spontaneous emulsion of water and oil on addition of a strong surface-active

agent. The term “microemulsion” was first used even later by Schulman et al. in 1959 to

describe a multiphase system consisting of water, oil, surfactant and alcohol, which forms a

transparent solution. There has been much debate about the word “microemulsion” to

describe such systems. Although not systematically used today, some prefer the names

“micellar emulsion” or “swollen micelles”. Microemulsions were probably discovered well

before the studies of Schulmann: Australian housewives have used since the beginning of last

century water/eucalyptus oil/soap flake/white spirit mixtures to wash wool, and the first

commercial microemulsions were probably the liquid waxes discovered by Rodawald in

1928. Interest in microemulsions really stepped up in the late 1970's and early 1980's when it

was recognized that such systems could improve oil recovery and when oil prices reached

levels where tertiary recovery methods became profit earning. Nowadays this is no longer the

case, but other microemulsion applications were discovered, e.g., catalysis, preparation of

submicron particles, solar energy conversion, liquid−liquid extraction (mineral, proteins,

etc.). Together with classical applications in detergency and lubrication, the field remains

sufficiently important to continue to attract a number of scientists. From the fundamental



research point of view, a great deal of progress has been made in the last 20 years in

understanding microemulsion properties. In particular, interfacial film stability and

microemulsion structures can now be characterized in detail owing to the development of

new and powerful techniques such as small-angle neutron scattering. The following sections

deal with fundamental microemulsion properties, i.e., formation and stability, surfactant

films, classification and phase behavior (Gelbart and BenShau, 1996).

Comparison between Emulsion & Microemulsion:

Property Emulsion Microemulsion

Composition Water, oil and emulsifier Water, oil, emulsifier and co-

surfactant

Appearance Cloudy Transparent

Viscosity High viscosity Low viscosity with Newtonian

behaviour

Particle size 1-20 µm 10-100 nm

Interfacial

tension

High Ultra low

Interfacial film Tough Highly Flexible

Manufacturing Tedious, high sheer needed Easy and spontaneous

Free energy Require large input of energy

during its preparation

Require low input of energy

during its preparation

Stability Thermodynamically unstable Thermodynamically stable

Cost Higher cost Relatively low cost



Figure 3: Micellar Solution, Microemulsion and Emulsion

Merits of Microemulsion-Based Systems:

Microemulsions exhibit several advantages as drug delivery systems (Surabhi et al.,

2010). They are as following:

Increases the rate of absorption

Eliminates variability in absorption

Helps solublize lipophilic drug

Provides a aqueous dosage form for water insoluble drugs

Increases bioavailability

Various routes like tropical, oral and intravenous can be used to deliver the product

Rapid and efficient penetration of the drug moiety.

Helpful in taste masking Provides protection from hydrolysis and oxidation as drug

in oil phase in o/w microemulsion is not exposed to attack by water and air.

Liquid dosage form increases patient compliance.



Less amount of energy requirement.

Demerits of Microemulsion-Based Systems:

The disadvantages of microemulsion are as follows (Bhowmik et al., 2013):

Use of a large concentration of surfactant and co-surfactant necessary for stabilizing

the nanodroplets.

Limited solubilizing capacity for high-melting substances.

The surfactant must be nontoxic for using pharmaceutical applications.

Microemulsion stability is influenced by environmental parameters such as

temperature and pH. These parameters change upon microemulsion delivery to

patients.

Types of Microemulion:

A well-known classification of microemulsions is that of Winsor who identified four

general types of phase equilibrium:

Type I (Winsor I):

The surfactant is preferentially soluble in water and oil-in-water (o/w) microemulsions

form (Winsor I). The surfactant-rich water phase coexists with the oil phase where

surfactant is only present as monomers at small concentration.

Type II (Winsor II):

The surfactant is mainly in the oil phase and water-in-oil (w/o) microemulsions form. The

surfactant-rich oil phase coexists with the surfactant-poor aqueous phase (Winsor II).

Type III (Winsor III):

It is a three-phase system where a surfactant-rich middle-phase coexists with both excess

water and oil surfactant-poor phases (Winsor III or middle-phase microemulsion).



Type IV (Winsor IV):

It is a single-phase (isotropic) micellar solution that forms upon addition of a sufficient

quantity of amphiphile (surfactant plus alcohol).

Figure 4: Types of Microemulsion

Depending on surfactant type and sample environment, types I, II, III or IV form

preferentially, the dominant type being related to the molecular arrangement at the

interface. Phase transitions are brought about by increasing either electrolyte

concentration (in the case of ionic surfactants) or temperature (for non-ionics). Various

investigators have focused on interactions in an adsorbed interfacial film to explain the

direction and extent of interfacial curvature. The first concept was that of Bancroft and

Clowes who considered the adsorbed film in emulsion systems to be duplex in nature,

with an inner and an outer interfacial tension acting independently .The interface would

then curve such that the inner surface was one of higher tension. Bancroft’s rule was

stated as “that phase will be external in which the emulsifier is most soluble”; i.e., oil-

soluble emulsifiers will form w/o emulsions and water-soluble emulsifiers o/w emulsions.

This qualitative concept was largely extended and several parameters have been proposed

to quantify the nature of the surfactant film (Jha et al., 2011).

Theories of Microemulsion Formation:



Historically, three approaches have been used to explain microemulsion formation and

stability. They are as follows:

Interfacial or mixed film theories.

Solubilization theories.

Thermodynamic treatments.

The free energy of microemulsion formation can be considered to depend on the extent to

which surfactant lowers the surface tension of the oil water interface and change in

entropy of the system such that,

Gf = γ a - T S

Where,

Gf = free energy of formation

A = change in interfacial area of microemulsion

S = change in entropy of the system

T = temperature

γ = surface tension of oil water interphase

It should be noted that when a microemulsion is formed the change in A is very large due

to the large number of very small droplets formed. In order for a microemulsion to be

formed (transient) negative value of was required, it is recognized that while value of is

positive at all times, it is very small and it is offset by the entropic component. The

dominant favorable entropic contribution is very large dispersion entropy arising from the

mixing of one phase in the other in the form of large number of small droplets. However

there are also expected to be favorable entropic contributions arising from other dynamic

processes such as surfactant diffusion in the interfacial layer and monomermicelle

surfactant exchange. Thus a negative free energy of formation is achieved when large

reductions in surface tension are accompanied by significant favorable entropic change.

In such cases, microemulsion is spontaneous and the resulting dispersion is

thermodynamically stable (Jha et al., 2011).



Formulation Aspects of Microemulsion:

Microemulsion Formulation:

Microemulsions are usually developed empirically since no adequate theory exists to

predict from which materials they are formed. Their formulation usually involves a

combination of three to five components; an oil phase, aqueous phase, primary surfactant,

secondary surfactant or cosurfactant and sometimes an electrolyte. These isotropic

systems are usually more difficult to formulate than ordinary emulsions because their

formation is a highly specific process involving spontaneous interactions amongst the

constituent molecules. Thus it is essential for a systematic study of microemulsion

composition to establish phase diagrams for the system under investigation. From these,

the extent of microemulsion region can be identified and its relation with other phases

established.The key to prepare microemulsion lies in controlling the curvature and

fluidity of the interfacial film that leads to a large increase of total interfacial area of the

system. Thus, the spontaneous formation of microemulsion can only occur when the

interfacial tension is so low that the increase in free energy of the system due to newly

created surfaces can be compensated by the increased entropy due to fluctuating

interfacial films and dispersion of the droplets. So, a relatively large amount of surfactant

is required to lower the interfacial tension in formulating conventional microemulsions. A

cosurfactant such as short-chain alcohols from C3 to C6 is often added to form

microemulsions (Taha et al., 2002).

Components of Microemulsion System:

A large number of oils and surfactant are available but their use in the microemulsion

formulation is restricted due to their toxicity, irritation potential and unclear mechanism

of action. Oils and surfactant which will be used for the formulation of microemulsion

should be biocompatible, non-toxic, clinically acceptable, and use emulsifiers in an

appropriate concentration range that will result in mild and non-aggressive

microemulsion. The emphasis is, excipients should be generally regarded as safe (GRAS)

(Talegaonkar et al., 2008).



Oil Phase:

The oil component influences curvature by its ability to penetrate and swell the tail group

region of the surfactant monolayer. As compare to long chain alkanes, short chain oil

penetrate the tail group region to a greater extent and resulting in increased negative

curvature (and reducedeffective HLB). Following are the different oils mainly used for

the formulation of microemulsion (Jha et al., 2011):

Saturated fatty acid-lauric acid,

Myristic acid, capric acid

Unsaturated fatty acid-oleic acid,

Linoleic acid, linolenic acid Fatty acid ester-ethyl or methyl esters of lauric,

Myristic and oleic acid.

The main criterion for the selection of oil is that the drug should have high solubility in it.

This will minimize the volume of the formulation to deliver the therapeutic dose of the

drug in an encapsulated form.

Surfactants

The role of surfactant in the formulation of microemulsion is to lower the interfacial

tension which will ultimately facilitates dispersion process during the preparation of

microemulsion and provide a flexible around the droplets. The surfactant should have

appropriate lipophilic character to provide the correct curvature at the interfacial region.

Generally, low HLB surfactants are suitable for w/o microemulsion, whereas high HLB

(>12) are suitable for o/w microemulsion. Following are the different surfactants are

mainly used for microemulsion (Jha et al., 2011):

Polysorbate (Tween 80 and Tween 20),

Lauromacrogol 300,

Lecithins, Decyl polyglucoside (Labrafil M 1944 LS),

Polyglyceryl-6-dioleate (Plurol Oleique),



Dioctyl sodium sulfosuccinate (Aersol OT), PEG-8 caprylic/capril glyceride

(Labrasol).

Cosurfactants:

Cosurfactants are mainly used in microemulsion formulation for following reasons: They

allow the interfacial film sufficient flexible to take up different curvatures required to

form microemulsion over a wide range of composition. Short to medium chain length

alcohols (C3-C8) reduce the interfacial tension and increase the fluidity of the interface.

Surfactant having HLB greater than 20 often require the presence of cosurfactant to

reduce their effective HLB to a value within the range required for microemulsion

formulation. Following are the different cosurfactant mainly used for microemulsion (Jha

et al., 2011):

Sorbitan monoleate,

Sorbitan monosterate,

Propylene glycol, propylene glycol monocaprylate (Capryol 90),

2-(2ethoxyethoxy) ethanol (Transcutol) and ethanol.

Preparation of Microemulsion:

Following are the different methods are used for the preparation of microemulsion:

Phase titration method

Phase inversion method

Phase Titration Method:

Microemulsions are prepared by the spontaneous emulsification method (phase titration

method) and can be portrayed with the help of phase diagram. As quaternary phase

diagram (four component system) is time consuming and difficult to interpret, pseudo

ternary phase diagram is constructed to find out the different zones including

microemulsion zone, in which each corner of the diagram represents 100% of the

particular components. Pseudoternary phase diagrams of oil, water, and

cosurfactant/surfactants mixtures are constructed at fixed cosurfactant/surfactant weight



ratios. Phase diagrams are obtained by mixing of the ingredients, which shall be

preweighed into glass vials and titrated with water and stirred well at room temperature.

Formation of monophasic/ biphasic system is confirmed by visual inspection. In case

turbidity appears followed by a phase separation, the samples shall be considered as

biphasic. In case monophasic, clear and transparent mixtures are visualized after stirring;

the samples shall be marked as points in the phase diagram. The area covered by these

points is considered as the microemulsion region of existence (Jha et al., 2011).

Figure 5: A Pseudo ternary Phase Diagram of Oil, Water & Surfactant Showing

Microemulsion Regions

Phase Inversion Method:

Phase inversion of microemulsion is carried out upon addition of excess of the dispersed

phase or in response to temperature. During phase inversion drastic physical changes

occur including changes in particle size that can ultimately affect drug release both in

vitro and in vivo. For non-ionic surfactants, this can be achieved by changing the

temperature of the system, forcing a transition from an o/w microemulsion at low

temperature to a w/o microemulsion at higher temperatures (transitional phase inversion).

During cooling, the system crosses a point zero spontaneous curvature and minimal

surface tension, promoting the formation of finely dispersed oil droplets. Apart from

temperature, salt concentration or pH value may also be considered. A transition in the

radius of curvature can be obtained by changing the water volume fraction. Initially water

droplets are formed in a continuous oil phase by successively adding water into oil.

Increasing the water volume fraction changes the spontaneous curvature of the surfactant

from initially stabilizing a w/o microemulsion to an o/w microemulsion at the inversion

locus (Jha et al., 2011).



Figure 6: A Tertiary Phase Diagram Portraying Various Structures of Microemulsion

Factors Affecting the Formation of Microemulsion:

The formation of microemulsion will depend on the following factors are:

Packing Ratio:

The HLB of surfactant determines the type of microemulsion through its influence on

molecular packing and film curvature. The analysis of film curvature for surfactant’s

association leadings to the formation of microemulsion, molecular packing and film

curvature. The analysis of film curvature for surfactant association’s leadings to the

formation of microemulsion (Ghosh et al., 2006).

Property of Surfactant, Oil Phase and Temperature:

The type of microemulsion depends on the nature of surfactant. Surfactant contains

hydrophilic head group and lipophilic tail group. The areas of these groups, which are a

measure of the differential tendency of water to swell head group and oil to swell the tail

area, are important for specific formulation when estimating the surfactant HLB in a



particular system. When a high concentration of the surfactant is used or when the

surfactant is in presence of salt, degree of dissociation of polar groups becomes lesser and

resulting system may be w/o type. Diluting with water may increase dissociation and

leads to an o/w system. Ionic surfactants are strongly influenced by temperature. It mainly

causes increased surfactant counter-ion dissociation. The oil component also influences

curvature by its ability to penetrate and hence swell the tail group region of the surfactant

monolayer. Short chains oils penetrate the lipophilic group region to a great extent and

results in increased negative curvature. Temperature is extremely important in

determining the effective head group size of nonionic surfactants. At low temperature,

they are hydrophilic and form normal o/w system. At higher temperature, they are

lipophilic and form w/o systems. At an intermediate temperature, microemulsion coexists

with excess water and oil phases and forms bicontinuous structure (Ghosh et al., 2006).

The Chain length, Type and Nature of Co-surfactant:

Alcohols are widely used as a co-surfactant in microemulsions. Addition of shorter chain

co-surfactant gives positive curvature effect as alcohol swells the head region more than

tail region so, it becomes more hydrophilic and o/w type is favored, while longer chain

co-surfactant favors w/o type w/o type by alcohol swelling more in chain region than

head region (Ghosh et al., 2006).

Characterization of Microemulsions:

The characterization of microemulsions is a difficult task due to their complexity, variety

of structures and components involved in these systems, as well as the limitations

associated with each technique but such knowledge is essential for their successful

commercial exploitation. Phase behavior studies are essential for the study of surfactant

system determined by using phase diagram that provide information on the boundaries of

the different phases as a function of composition variables and temperatures, and, more

important, structural organization can be also inferred. Phase behaviour studies also

allow comparison of the efficiency of different surfactants for a given application. In the

phase behaviour studies, simple measurement and equipments are required. The

boundaries of one-phase region can be assessed easily by visual observation of samples of

known composition. The main drawback is long equilibrium time required for

multiphase region, especially if liquid crystalline phase is involved (Martin et al., 1994).



Scattering Techniques for Microemulsions Characterization:

Small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), and static

as well as dynamic light scattering are widely applied techniques in the study of

microemulsions. These methods are very valuable for obtaining quantitative information

on the size, shape and dynamics of the components. The major drawback of this

technique is the dilution of the sample required for the reduction of interparticular

interaction. This dilution can modify the structure and the composition of the

pseudophases. Nevertheless, successful determinations have been carried out using a

dilution technique that maintains the identity of droplets. Small-angle X-ray scattering

techniques have been used to obtain information on droplet size and shape (Regev et al.,

1996).

Nuclear Magnetic Resonance Studies:

The structure and dynamics of microemulsions can be studied by using nuclear magnetic

resonance techniques. Self-diffusion measurements using different tracer techniques,

generally radio labeling, supply information on the mobility of the components. The

Fourier transform pulsed-gradient spin-echo (FT-PGSE) technique uses the magnetic

gradient on the samples and it allows simultaneous and rapid determination of the

selfdiffusion coefficients (in the range of 10-9 to 10-12 m2s-1), of many components

(Shinoda et al., 1991).

Interfacial Tension:

The formation and the properties of microemulsion can be studied by measuring the

interfacial tension. Ultra low values of interfacial tension are correlated with phase

behavior, particularly the existence of surfactant phase or middle-phase microemulsions

in equilibrium with aqueous and oil phases. Spinning-drop apparatus can be used to

measure the ultra low interfacial tension. Interfacial tensions are derived from the

measurement of the shape of a drop of the low-density phase, rotating it in cylindrical

capillary filled with high-density phase (Vyas et al., 2002).

Viscosity Measurements:

Viscosity measurements can indicate the presence of rod-like or worm-like reverse

micelle. Viscosity measurements as a function of volume fraction have been used to



determine the hydrodynamic radius of droplets, as well as interaction between droplets

and deviations from spherical shape by fitting the results to appropriate models (e.g. for

microemulsions showing Newtonian behavior, Einstein’s equation for the relative

viscosity can be used to calculate the hydrodynamic volume of the particles) (bellare et

al., 1999).

Electron Microscope Characterization:

Transmission Electron Microscopy (TEM) is the most important technique for the study

of microstructures of microemulsions because it directly produces images at high

resolution and it can capture any co-existent structure and micro-structural transitions.

There are two variations of the TEM technique for fluid samples.

The cryo-TEM analyses in which samples are directly visualized after fast freeze and

freeze fructose in the cold microscope.

The Freeze Fracture TEM technique in which a replica of the specimen is images

under RT conditions.

Evaluation of Microemulsion:

The microemulsions are evaluated by the following techniques. They are:

Parameters Techniques Used

Phase Behaviour Phase contrast microscopy &freeze fracture TEM

Size and Shape Transmission Electron Microscopy(TEM), SEM

Rheology Brookfield viscometer

Conductivity Conductivity meter

Zeta potential Zetasizer

pH pH meter

Drug release studies Franz Diffusion Cells

Physical stability

study

Ultracentrifuge



Phase Behavior Studies:

Visual observations, phase contrast microscopy and freeze fracture transmission electron

microscopy can be used to differentiate microemulsions from liquid crystals and coarse

emulsions. Clear isotropic one-phase systems are identified as microemulsions whereas

opaque systems showing bifringence when viewed by cross polarized light microscopy

may be taken as liquid crystalline system (Katiyar et al., 2013).

Rheology:

Change in the rheological characteristics help in determining the microemulsion region

and its separation from other related structures like liquid crystals. Bicontinuous

microemulsion are dynamic structures with continuous fluctuations occurring between the

Bicontinuous structure, swollen reverse micelle, and swollen micelles (Katiyar et al.,

2013).

Scattering Techniques:

Scattering techniques such as small angle neutron scattering, small angle X-ray scattering

and light scattering have found applications in studies of microemulsion structure,

particularly in case of dilute monodisperese spheres, when polydisperse and/or

concentrated systems such as those frequently seen in microemulsions (Katiyar et al.,

2013).

Applications of Microemulsion:

During the last two decades, microemulsions have been promisingly used as drug

delivery system for its advantages include their thermodynamic stability, optical clarity

and ease of penetration. The role of microemulsion as drug delivery system shall be

discussed herein.


route of administration of biotech products

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