CONTROLLED CONTROLLED DELIVERY OF DELIVERY OF

VACCINEVACCINE

CONTROLLED DELIVERY CONTROLLED DELIVERY OF VACCINE USING OF VACCINE USING

BIODEGRABLE SUBSTANCEBIODEGRABLE SUBSTANCE

ORIGIN OF VACCINEORIGIN OF VACCINE Smallpox was the first disease which are Smallpox was the first disease which are

inoculated with other type of type inoculated with other type of type infections which was believed to be infections which was believed to be started in India and China before 200BCstarted in India and China before 200BC

Scientists who are involved areScientists who are involved are 1) Lady Mary Worltey Montague1) Lady Mary Worltey Montague 2) Edward Jenner 2) Edward Jenner

3) Sarah Nelmes3) Sarah Nelmes

4) James Phipps4) James Phipps

DEVELOPMENT OF NEW DEVELOPMENT OF NEW VACCINEVACCINE

1998 first vaccine for 1998 first vaccine for rotavirus was developed;rotavirus was developed;

2121stst century in 2006 century in 2006 human papillomavirus was human papillomavirus was developed for cancer.developed for cancer.

DEFINATION OF VACCINEDEFINATION OF VACCINE Vaccine which are Vaccine which are

suspensions of killed, lived, suspensions of killed, lived, or attenuated (having or attenuated (having weakened virulence) weakened virulence) cultures of micro-organism cultures of micro-organism are used as antigen to are used as antigen to produce immunity against produce immunity against infection due to the infection due to the particular microorganism particular microorganism

ExampleExample→→ typhoid fever typhoid fever vaccine consists of killed vaccine consists of killed cell salmonella typhii cell salmonella typhii CHOLREACHOLREA

TYPE OF VACCINE TYPE OF VACCINE INACTIVATED VACCINE- Microorganism that has INACTIVATED VACCINE- Microorganism that has

been killed chemicals or heat. This type of been killed chemicals or heat. This type of vaccine has less immune response due to which vaccine has less immune response due to which booster doses require.booster doses require.

Example – vaccine against flu, choleraExample – vaccine against flu, cholera2) Live attenuated – These are live microorganism 2) Live attenuated – These are live microorganism

that has been cultivated under condition which that has been cultivated under condition which disables their virulent properties. They have disables their virulent properties. They have durable immunological reponsedurable immunological reponse

Example Example →→yellow fever, measles, rubella and yellow fever, measles, rubella and mumpsmumps

CLASSIFICATION OF COMMON CLASSIFICATION OF COMMON USED VACCINEUSED VACCINE

DISEASE DISEASE TYPE OF TYPE OF VACCINEVACCINE

Tuberculosis Tuberculosis Attenuated Attenuated

Cholera Cholera Inactivated Inactivated

Polio Polio Attenuated Attenuated

Influenza Influenza Inactivated Inactivated

Diphtheria Diphtheria Inactivated Inactivated exotoxinexotoxin

H influenza typeH influenza type Polysaccharide+Polysaccharide+proteinprotein

IMMUNIZATION SCHEDULE FOR IMMUNIZATION SCHEDULE FOR CHILDREN IN INDIACHILDREN IN INDIA

AGE OF CHILDAGE OF CHILD VACCINEVACCINE

1& ½ 1& ½ BCG, DPT*1 AND BCG, DPT*1 AND PILIO*1PILIO*1

2 & ½ 2 & ½ DPT*2 & PILIO*2DPT*2 & PILIO*2

3 & ½ 3 & ½ DPT*3 & PILIO*3DPT*3 & PILIO*3

9 months9 months Measles Measles

Below 16 and 24 Below 16 and 24 monthsmonths

DPT*4 & PILIO*4DPT*4 & PILIO*4

DEVELOPMENT AND DESIGNING DEVELOPMENT AND DESIGNING OF NEW GENERATION VACCINEOF NEW GENERATION VACCINE

1)1) Multivalent subunit Multivalent subunit vaccinevaccine

(a)Small matrix antibody (a)Small matrix antibody antigen complexesantigen complexes

(b) Liposomes (b) Liposomes

2)Purified 2)Purified macromolecules macromolecules

3)Synthetic peptides as 3)Synthetic peptides as vaccine vaccine

4) Immunoadhesions 4) Immunoadhesions

5) Antigen vaccine5) Antigen vaccine

(a) Recombinant (a) Recombinant vaccinesvaccines

6) Vector vaccines 6) Vector vaccines

(a)(a) Recombinant vector Recombinant vector vaccinevaccine

7) Anti idiotype vaccines7) Anti idiotype vaccines

8) Targeted immune 8) Targeted immune stimulantsstimulants

OBJECTIVE OF THE VACCINE OBJECTIVE OF THE VACCINE DELIVERYDELIVERY

To elicit a protective immune response of To elicit a protective immune response of sufficiently long duration, from a single-sufficiently long duration, from a single-contact immunization contact immunization

Potentiate the immune response to Potentiate the immune response to vaccine without manifesting any adverse vaccine without manifesting any adverse effects on the bodyeffects on the body

Incorporate many in a single formulation Incorporate many in a single formulation deliver even those vaccines through the deliver even those vaccines through the oral route that currently need to be given oral route that currently need to be given parenterally. parenterally.

INDUCTION OF IMMUNE INDUCTION OF IMMUNE RESPONSERESPONSE

Vaccine is carried out to protect the Vaccine is carried out to protect the individual from by priming the immune individual from by priming the immune system to resist the infecting agent. system to resist the infecting agent.

This resistance can be offered by effectors This resistance can be offered by effectors molecules antibodies, cytokines, molecules antibodies, cytokines, complement etc or effectors cell (cell complement etc or effectors cell (cell mediated immunity).mediated immunity).

Immunization also results in the system Immunization also results in the system developing a “memory” of exposure to developing a “memory” of exposure to antigen present on the pathogenantigen present on the pathogen

HOW IMMUNE RESPONSE IS HOW IMMUNE RESPONSE IS GENERATED ?GENERATED ?

Immune response Immune response

1) Cell type immune response1) Cell type immune response

a) Non specific cell type a) Non specific cell type

i) Phagocytes i) Phagocytes

ii) Auxiliary cellii) Auxiliary cell

b) Targeted specific cell type b) Targeted specific cell type

i) B celli) B cell

ii) T cell ii) T cell

2) Effector response2) Effector response

3) Humoral immunity3) Humoral immunity

4) Cell mediated immunity4) Cell mediated immunity

MECHANISM ACTION OF THE MECHANISM ACTION OF THE CELL TYPE IMMUNE RESPONSECELL TYPE IMMUNE RESPONSE

Phagocytes and auxiliary are Phagocytes and auxiliary are first line defence against first line defence against infection infection

Inflammatory substance Inflammatory substance released from auxiliary cell on released from auxiliary cell on encounter with the foreigen encounter with the foreigen material, stimulate phagocytes material, stimulate phagocytes and attract from the site of and attract from the site of infection.infection.

These cell get engulf the These cell get engulf the material upon the phagocytesmaterial upon the phagocytes

Fragments of proteins produce Fragments of proteins produce are essential for generation of a are essential for generation of a T cellT cell

While performing the function While performing the function macrophage act as antigen macrophage act as antigen presenting cell (APC)presenting cell (APC)

The carry metabolic organelles The carry metabolic organelles to kill micro-organism by to kill micro-organism by generating the free radicalgenerating the free radical

MECHANISM OF MECHANISM OF IMMUNOGENCITY BY CARRIER IMMUNOGENCITY BY CARRIER

MOLECULESMOLECULES The coupling of small peptides to carrier proteins The coupling of small peptides to carrier proteins

increase the molecular mass of peptide and there increase the molecular mass of peptide and there by improves the uptake by APCby improves the uptake by APC

Biological half of small peptides may be Biological half of small peptides may be increased via the linkage to carrier proteinincreased via the linkage to carrier protein

If the synthetic peptides itself is immunogenic If the synthetic peptides itself is immunogenic (as in the case of peptides having B cell and T (as in the case of peptides having B cell and T cell epitopes ), the carriers proteins merely cell epitopes ), the carriers proteins merely functions as a polymeric delivery system.functions as a polymeric delivery system.

MECHANISM OF MECHANISM OF IMMUNOGENICITY VIE CARRIER IMMUNOGENICITY VIE CARRIER

MOLECULESMOLECULES Commonly used Commonly used

carrier are carrier are

1)1) Keyholes limpet Keyholes limpet hemocyin (KLH) and hemocyin (KLH) and sperm whale sperm whale myoglobin (SWM)myoglobin (SWM)

2)2) Albumins Albumins (ovalbumins)(ovalbumins)

3)3) Fig will show the Fig will show the attachment of attachment of hapten to carrier hapten to carrier moleculesmolecules

EFFECTOR RESPONSE EFFECTOR RESPONSE Effectors response of the T cell consist of secreting Effectors response of the T cell consist of secreting

specialized substance called cytokines. specialized substance called cytokines. Cytokines are divided into two broad classCytokines are divided into two broad class1)1) Helper T cell help B & T cell in variety of ways.Helper T cell help B & T cell in variety of ways.2)2) Cytokines or “killer” T cell make the cytokines that killed Cytokines or “killer” T cell make the cytokines that killed

the infected cellsthe infected cells Antibodies produce during this process are help in the in Antibodies produce during this process are help in the in

binding with invading microorganism and facilitating the binding with invading microorganism and facilitating the pathogens bearing the Ag phagocytes.pathogens bearing the Ag phagocytes.

T cell are divided into TH1 which are primary involved in T cell are divided into TH1 which are primary involved in cell mediated immunity and TH 2 with the production of cell mediated immunity and TH 2 with the production of antibodiesantibodies

►►T cell are divided T cell are divided into TH1 which are into TH1 which are primary in cell primary in cell mediated and TH 2 mediated and TH 2 with the production with the production

►►TH 1 secrete TH 1 secrete gamma interferon gamma interferon TNF TNF

► ►TH2 secrete IL-2, TH2 secrete IL-2, IL-5,IL-6, IL-10IL-5,IL-6, IL-10

CELL MEDIATED IMMUNITYCELL MEDIATED IMMUNITY On the exposure to the appropriate antigen T On the exposure to the appropriate antigen T

lymphocytes of lymphoid tissue proliferated to lymphocytes of lymphoid tissue proliferated to form activated T cell which are of three type form activated T cell which are of three type

1) Helper T cell – release lymphokines which 1) Helper T cell – release lymphokines which stimulate growth and differentiate of the stimulate growth and differentiate of the activated B cell to from plasma cellactivated B cell to from plasma cell

2)Cytotoxic T cell which bind tightly to specific 2)Cytotoxic T cell which bind tightly to specific binding antigen which then form secrete pore and binding antigen which then form secrete pore and form round role in the membraneform round role in the membrane

3) Suppresser T cell tthey are believed to inhibit the 3) Suppresser T cell tthey are believed to inhibit the conversion of B cell into plasma cellconversion of B cell into plasma cell

DESIGING RATIONALE AND DESIGING RATIONALE AND REQUIRMENT OF AN EFFICIENT REQUIRMENT OF AN EFFICIENT

VACCINE DELIVERYVACCINE DELIVERY Particulate AntigenParticulate Antigen Booster DosesBooster Doses Adjuvant – commonly used is Adjuvant – commonly used is

Calcium phosphateCalcium phosphate

● ● Antigen Stability Antigen Stability

PARTICULATE ANTIGENPARTICULATE ANTIGEN Uptake is generally take by phagocytes which are Uptake is generally take by phagocytes which are

important for eliciting an immune response: the important for eliciting an immune response: the vaccine formulation should be amenable to vaccine formulation should be amenable to phagocytosis.phagocytosis.

Soluble material which can be moved about Soluble material which can be moved about anywhere is taken up by the pinocytosis. which is anywhere is taken up by the pinocytosis. which is 10 times less efficiency than phagocytosis10 times less efficiency than phagocytosis

The efficiency of uptake can make the The efficiency of uptake can make the formulation in a particulate form.formulation in a particulate form.

Finally particulate matter is important signal for Finally particulate matter is important signal for phagocytic cells to home towards the sitephagocytic cells to home towards the site

BOOSTER DOSESBOOSTER DOSES

Vaccine that are required to be given in Vaccine that are required to be given in the divided doses to produce the more the divided doses to produce the more efficiency efficiency

High doses tolerance is another curious High doses tolerance is another curious phenomenon which occur if size of the phenomenon which occur if size of the doses is too high which rendered the doses is too high which rendered the antigen and no response is producedantigen and no response is produced

Efficiency of the divide doses can be Efficiency of the divide doses can be sought with the reference to cellular sought with the reference to cellular events that takes place in the events that takes place in the immunizationimmunization

ADJUVANTS ADJUVANTS Vaccine formulation is usually administered with Vaccine formulation is usually administered with

the special additive called “adjuvant “ special the special additive called “adjuvant “ special when the vaccine is in the form of killed when the vaccine is in the form of killed pathogens or isolated protein instead of live pathogens or isolated protein instead of live attenuated organism”attenuated organism”

“ “ A VACCINE DELIVERY SYSTEM IS ALSO A VACCINE DELIVERY SYSTEM IS ALSO EXPECTED TO HAVE SUFFICIENT ADJUVANTTICITY EXPECTED TO HAVE SUFFICIENT ADJUVANTTICITY TO POTENTIATE THE REPONSE TO DELIVERY TO POTENTIATE THE REPONSE TO DELIVERY ANTIGEN.” ANTIGEN.”

Commonly used adjuvant are calcium phosphate Commonly used adjuvant are calcium phosphate which act as adjuvant and improved safety profile which act as adjuvant and improved safety profile and enhance immune system stimulationand enhance immune system stimulation

PRODUCTION OF CAPPRODUCTION OF CAP

♦♦The formulation of CAP nanoparticles is easily tailored for The formulation of CAP nanoparticles is easily tailored for each antigen.each antigen. The manufacturing process is quick and requires only The manufacturing process is quick and requires only

simple equipment, water for injection, and inorganic simple equipment, water for injection, and inorganic salts.salts.

The CAP nanoparticle have exceptional protein loading The CAP nanoparticle have exceptional protein loading capacity: about 20%(w/w) if antigen is coated on the capacity: about 20%(w/w) if antigen is coated on the surfaces only and about 50% and greater for internal surfaces only and about 50% and greater for internal “core loading”.“core loading”.

Batch-to batch consistency is excellent. Batch-to batch consistency is excellent. Long-term storage (out to one year) resulted in no Long-term storage (out to one year) resulted in no

changes in particles size, ph and surface morphology changes in particles size, ph and surface morphology

ANTIGEN STABILITYANTIGEN STABILITY Antigen stability – antigen used for Antigen stability – antigen used for

immunization is subjected to immunization is subjected to environmental stress such as temperature environmental stress such as temperature ph or non-aqueous me3dium which may ph or non-aqueous me3dium which may cause the change confirmation.cause the change confirmation.

This may result the production of epitopes This may result the production of epitopes which are meant for generate the B cell which are meant for generate the B cell responseresponse

Requirement for maintaining the stability Requirement for maintaining the stability of the antigen are follows – low of the antigen are follows – low temperatures but not freezing, physilogical temperatures but not freezing, physilogical ph and aqueous environment ph and aqueous environment

PREPARATION OF PREPARATION OF MICROSPHERE IN CONTROLLED MICROSPHERE IN CONTROLLED

DELIVERYDELIVERY The properties for preparation of The properties for preparation of

microsphere are microsphere are 1) optimal antigen loading1) optimal antigen loading 2) size of microsphere 2) size of microsphere 3) minimum wastage of material 3) minimum wastage of material 4) within batch uniformity and inter-batch 4) within batch uniformity and inter-batch

reproducibilityreproducibility 5) minimum exposures of Ag to denaturing 5) minimum exposures of Ag to denaturing

conditioncondition6) For parentrally products microsphere 6) For parentrally products microsphere

need to be sterileneed to be sterile

POLY ( LACTIDE-CO-GLYCIDE)POLY ( LACTIDE-CO-GLYCIDE)(PLGA)(PLGA)

This type of the microsphere which is used for the This type of the microsphere which is used for the controlled delivery system of peptides, native and controlled delivery system of peptides, native and synthetic proteins and lately.synthetic proteins and lately.

PLGA microsphere are composed of a spherical PLGA microsphere are composed of a spherical shaped polymeric matrix ranging in diameter shaped polymeric matrix ranging in diameter from 1 to 250 micrometere.from 1 to 250 micrometere.

Many factors are important to formulate Many factors are important to formulate i) Ability to release the entrapped substancei) Ability to release the entrapped substance ii) Particle sizeii) Particle size iii) Stability and safety which is related to in vivo iii) Stability and safety which is related to in vivo

polymer degrationpolymer degration

Antigen are physically entrapped into Antigen are physically entrapped into microsphere inject able solid polymeric matrixmicrosphere inject able solid polymeric matrix

The combination of diffusion through pores and of The combination of diffusion through pores and of polymer matrix biodegradable allows the control polymer matrix biodegradable allows the control of antigen release ratesof antigen release rates

The biodegrading rates of polymer depends on its The biodegrading rates of polymer depends on its molecular weightmolecular weight

After hydrolysis degraded products form After hydrolysis degraded products form monomer of lactide and glycolide which are monomer of lactide and glycolide which are eliminated by Krebs cycle as carbon dioxide & eliminated by Krebs cycle as carbon dioxide & urine.urine.

During the process, the encapsulated antigen are During the process, the encapsulated antigen are release which can varies from hours to months release which can varies from hours to months depending upon the polymer combinationdepending upon the polymer combination

Microsphere contdMicrosphere contd Microsphere diameter plays an important role in Microsphere diameter plays an important role in

interaction with phagocytic cellsinteraction with phagocytic cells Particle smaller than the 10 micrometer Particle smaller than the 10 micrometer

phagocytosed faster by the macrophage which phagocytosed faster by the macrophage which cause recruitment to the site of administration cause recruitment to the site of administration after subcutaneous injection.after subcutaneous injection.

Particle more than 10 micrometer act as depot Particle more than 10 micrometer act as depot releasing the antigen. releasing the antigen.

Polymer selection also plays an important role in Polymer selection also plays an important role in the manufactures because it critically influence the manufactures because it critically influence their rate of biodegrading their rate of biodegrading

Contd Contd By changing the homopolymer By changing the homopolymer ratio different physicochemical ratio different physicochemical

compositions can influence its compositions can influence its degradability and permeabilitydegradability and permeability

These parameter can be easily These parameter can be easily monitored using different monitored using different polymer and copolymerpolymer and copolymer

Amorphous polymer are more Amorphous polymer are more permeable than PLGApermeable than PLGA

The combination of particles The combination of particles with different compositions in with different compositions in the same formulation has the same formulation has permitted to establish the permitted to establish the concept of the single dose concept of the single dose vaccinevaccine

MICROSPHERE PERPARATIONMICROSPHERE PERPARATION

This can be done by two methods This can be done by two methods 1) Simple emulsification (O\W)1) Simple emulsification (O\W) 2) Multiple water–in-oil-water (W\O\W)2) Multiple water–in-oil-water (W\O\W)♦ ♦ the choice of method depends on the physical and chemical the choice of method depends on the physical and chemical

characteristics of antigen, permitting the matrix entrapment of both characteristics of antigen, permitting the matrix entrapment of both lipophilic and hydrophiliclipophilic and hydrophilic

♦ ♦ in the first case an emulsion is formed by dissolving the antigen in an in the first case an emulsion is formed by dissolving the antigen in an organic solvent immiscible in water (methlyene chlorides ) organic solvent immiscible in water (methlyene chlorides ) containing the polymer , under strong mechanical agitation .containing the polymer , under strong mechanical agitation .

this emulsion can be stabilized by added surfactants to the aqueous this emulsion can be stabilized by added surfactants to the aqueous phase.phase.

The solvent is eliminated by the evaporation at room temperatures, The solvent is eliminated by the evaporation at room temperatures, followed by the washed and freezing dryingfollowed by the washed and freezing drying

CONTDCONTD The multiple emulsion method involves water-in-oil-in-water The multiple emulsion method involves water-in-oil-in-water

emulsificationemulsification The inner aqueous phase containing the hydrophilic The inner aqueous phase containing the hydrophilic

substance is obtained after emulsification with the substance is obtained after emulsification with the immiscible organic solvent containing polymer under strong immiscible organic solvent containing polymer under strong mechanical agitation.mechanical agitation.

This emulsion is stabilized by the addition of aqueous This emulsion is stabilized by the addition of aqueous solution containing a surfactants (e.g. poly vinyl alcohol ) solution containing a surfactants (e.g. poly vinyl alcohol ) and is further homogenized to produce a W\O\W double and is further homogenized to produce a W\O\W double emulsion.emulsion.

This double emulsion is gently stirred with homogenizer This double emulsion is gently stirred with homogenizer room temperatures for solvent evaporation room temperatures for solvent evaporation

The microsphere are collected by the centrifugation, The microsphere are collected by the centrifugation, washed with water and freeze dried.washed with water and freeze dried.

the mixture of hydrphilic and lipophilic molecules can be the mixture of hydrphilic and lipophilic molecules can be successfully entrapped by this methodsuccessfully entrapped by this method

PGLA MICROSPHERE PGLA MICROSPHERE BIOCOMPATIBLITYBIOCOMPATIBLITY

Polymer microspheres have attracted much attention because of their Polymer microspheres have attracted much attention because of their biocompatible characteristics the phagocytosis of biodegradable and biocompatible characteristics the phagocytosis of biodegradable and no biodegradable particles has reported to depend on their size, surface no biodegradable particles has reported to depend on their size, surface charge and hydrophobicity.charge and hydrophobicity.♦♦ After subcutaneous administration, PLGA microsphere ranging in the After subcutaneous administration, PLGA microsphere ranging in the diameter from 1 to 10 micrometer is readily phagocytosed by diameter from 1 to 10 micrometer is readily phagocytosed by macrophages recruited to the site of injection, thereby providing an macrophages recruited to the site of injection, thereby providing an intracellular delivery of the antigen. intracellular delivery of the antigen. ♦♦This mechanism may enhance antibody response and consequently This mechanism may enhance antibody response and consequently decreases the required antigen dose. On the other hand, particles larger decreases the required antigen dose. On the other hand, particles larger than 10 micrometer in diameter would remain as a depot at the site of than 10 micrometer in diameter would remain as a depot at the site of injection providing a sustained release of antigen.injection providing a sustained release of antigen. Improving their hydrophobicity can increase phagocytosis of the particles, Improving their hydrophobicity can increase phagocytosis of the particles, whereas microsphere preparation with different compositions of PLA and whereas microsphere preparation with different compositions of PLA and PLGA do alter the extent of phagocytosis. PLGA do alter the extent of phagocytosis.

ROUTE OF IMMUNIZATIONROUTE OF IMMUNIZATION The oral route of delivery is the most acceptable in terms of patient The oral route of delivery is the most acceptable in terms of patient

compliance.compliance. the antigen entering the body through the oral route include items of the antigen entering the body through the oral route include items of

food, and are “tolergenic”.food, and are “tolergenic”. Lymphoid tissue associated with the digestive tract is located primarily Lymphoid tissue associated with the digestive tract is located primarily

in Peyer’s patches in the small intestine and the material that targets in Peyer’s patches in the small intestine and the material that targets to Peyer’s patches often evokes an immune response. to Peyer’s patches often evokes an immune response.

This response is often in a such type as protect against pathogens This response is often in a such type as protect against pathogens entering the body by invading mucosal tissue if the response against entering the body by invading mucosal tissue if the response against microbes invading by non-mucosal routes is desired after oral microbes invading by non-mucosal routes is desired after oral immunization, the delivery system must be capable of transporting the immunization, the delivery system must be capable of transporting the

Ag to deep sealed lymphoid tissue instead of releasing it to the Ag to deep sealed lymphoid tissue instead of releasing it to the mucosa-associated lymphoid tissue.mucosa-associated lymphoid tissue.

Systemic routes of immunization should seek a route that ensures Systemic routes of immunization should seek a route that ensures maximal exposures of the Ag to immunocytes.maximal exposures of the Ag to immunocytes. The usual choices are intramuscular (i.m.) subcutaneous or The usual choices are intramuscular (i.m.) subcutaneous or interdermal immunization. interdermal immunization.

As the site delivery moves from deep As the site delivery moves from deep tissues towards superficial, the tissues towards superficial, the delivery system is exposed to a larger delivery system is exposed to a larger number of first lines defence cell (i.m, number of first lines defence cell (i.m, i.d) at the same time, the period of i.d) at the same time, the period of residence of the delivery system in the residence of the delivery system in the body decrease as the site of delivery body decrease as the site of delivery moves towards superficial tissuemoves towards superficial tissue..

Route of immunization also takes place Route of immunization also takes place by two way 1) Systemic immunizationby two way 1) Systemic immunization

2) Mucosal immunization2) Mucosal immunization

CONCLUSIONCONCLUSION

PLGA microsphere with an incorporated PLGA microsphere with an incorporated antigen represents a good antigen delivery antigen represents a good antigen delivery system for both cellular and humoral system for both cellular and humoral response.response.

The easy manufactures of microsphere The easy manufactures of microsphere and the possibility of administration by and the possibility of administration by different routes offer the additional different routes offer the additional advantage of their use as a advantage of their use as a pharmaceutically acceptable adjuvant for pharmaceutically acceptable adjuvant for vaccines.vaccines.

INSTRUMENTAL METHOD OF INSTRUMENTAL METHOD OF PERPARATIONPERPARATION

This method include This method include 1)1) Spray dryingSpray drying2)2) Air suspension coatingAir suspension coating3)3) Press grindingPress grinding4)4) Coaceration-Phase separationCoaceration-Phase separation i) Emulsification methodsi) Emulsification methods a) Oil-in-oil emulsion a) Oil-in-oil emulsion b) Oil-in-water emulsionb) Oil-in-water emulsion c) Multiple emulsionc) Multiple emulsion5) Solvent extraction 5) Solvent extraction 6) Rotary evaporations 6) Rotary evaporations

SPRAYING DRYINGSPRAYING DRYING In this method spray dried protein In this method spray dried protein

is suspended in a solution of the is suspended in a solution of the polymer in an organic solvent polymer in an organic solvent (methylene chloride (methylene chloride ortetrahydronfuran).ortetrahydronfuran).

1)This is then pumped into spray drier 1)This is then pumped into spray drier by a peristaltic pump.by a peristaltic pump.

2) Dry air at high pressure and inlet 2) Dry air at high pressure and inlet air temperature of 37ºC atomizes air temperature of 37ºC atomizes the air suspension, and the the air suspension, and the polymer forms a matrix entrapping polymer forms a matrix entrapping the protein as the solvent the protein as the solvent evaporates from the droplets. evaporates from the droplets.

Spherical particles of the desired Spherical particles of the desired size range and loading can thus size range and loading can thus preparedprepared..

DISADVANTAGE OF THIS DISADVANTAGE OF THIS METHOD METHOD

This method is relatively This method is relatively large batch size required for large batch size required for processing in view of the processing in view of the limitation of the limitation of the availableequipment.availableequipment.

♦ ♦ Even small volumes spray Even small volumes spray driers are not designed to driers are not designed to handle volumes of the order of handle volumes of the order of a few ml, which would be a few ml, which would be sufficient to produce sufficient to produce thousands of doses. thousands of doses.

This size dispersion of This size dispersion of microsphere produced by this microsphere produced by this method is also exposed to be method is also exposed to be large.large.

EVALUATION OF VACCINE EVALUATION OF VACCINE LAODED MICROSPHERELAODED MICROSPHERE

The evalulation factors involve The evalulation factors involve

1)Praticle size1)Praticle size

2) Antigen loading2) Antigen loading

3) Structural integrity of 3) Structural integrity of encapsulated antigenencapsulated antigen

4)Studies on the antigen release in 4)Studies on the antigen release in vitrovitro

5) Antigencity of the preparation5) Antigencity of the preparation

Praticle sizePraticle size Particles size can be easily Particles size can be easily

determine using light microscopy or determine using light microscopy or in the greater detail by scanning the in the greater detail by scanning the electron microscopy (SEM) which electron microscopy (SEM) which shows the scanning electron shows the scanning electron microphotography of abatch of the microphotography of abatch of the

microsphere.microsphere. SEM also helps in accessing SEM also helps in accessing

surface morphology of the surface morphology of the microsphere.microsphere.

Standards equipment for Standards equipment for micromerities such as Coulter micromerities such as Coulter Counter or laser diffractometers may Counter or laser diffractometers may also helop to determine the particle also helop to determine the particle size and size and

distributionsdistributions Laser based equipment employing Laser based equipment employing

the assessment of sedimentation the assessment of sedimentation rates, also referred to as “time of rates, also referred to as “time of transition” methodology is also used transition” methodology is also used

Studies on the antigen release in Studies on the antigen release in vitrovitro

In vitro Ag release studies In vitro Ag release studies have served as quality control have served as quality control parameters as well as index of parameters as well as index of the duration of immune the duration of immune response. response.

These help to understand the These help to understand the nature of release and provide nature of release and provide significant insight into the significant insight into the formulation variablesformulation variables

By dispersing the microsphere By dispersing the microsphere in a suitable buffer in a vial in a suitable buffer in a vial and subjecting it to stirring at and subjecting it to stirring at 37ºc, over the period of time.37ºc, over the period of time.

Samples were withdrawn out Samples were withdrawn out periodically and analyses for periodically and analyses for Ag release using suitable Ag release using suitable method. method.

NOVEL DRUG SYSTEMNOVEL DRUG SYSTEM

Novel drug system include following pointNovel drug system include following point1)Particulate delivery system 1)Particulate delivery system i) Liposomei) Liposome ii) Emulsionii) Emulsion iii) Micro sphere iii) Micro sphere 2)Cochleates2)Cochleates3)Mucoadhesive polymer3)Mucoadhesive polymer4)DNA vaccination4)DNA vaccination5)Transgenic plants an edible immunogen concept 5)Transgenic plants an edible immunogen concept