Vaccine drug delivery system

VACCINE DRUG DELIVERY
SYSTEM
Prepared by, M. PHARM (PHARMACEUTICS )
1. MAHARNAB PRADHAN (VP21PHAR0100005)
2. AMAN CHOUDHARY (VP21PHAR0100008)
3. PIYUSH BARAI (VP21PHAR0100010)
4. SHUBHAM YADAV (VP21PHAR0100006)
Under the guidance of,
Dr. G.V. Radha

Contents
• What is vaccines?
• History of vaccines
• Mechanism of vaccine
• Uptake of Antigens
• Single shot vaccines
• Mucosal vaccine delivery systems
• Transdermal vaccine delivery system

What is vaccines?
 Vaccines are biological preparation which provide active acquired immunity
against particular diseases.
 Vaccine word is derived from Latin word “Variolae vaccinea” (cowpox).
 It is made of disease causing microbes, which are killed or present in attenuated
form or it’s toxins or one of it’s surface proteins.
 It stimulates the body immune system against the microbe and destroy it.
 The administration of vaccine is called vaccination.

History of vaccine
• Edward Jenner developed 1st vaccine against small pox at 1798 from
cowpox.
• Louis pasture developed live attenuated cholera vaccine and inactivated
anthrax vaccine in 1897 and 1904 respectively.
• In 1923, Alexander Glenny introduce a method to inactivate tetanus
toxins, this method was used to developed diphtheria vaccine in 1926.
• Viral tissue culture method was developed in 1950-1985, which helped in
development of inactivated and live attenuated polio vaccines.

Important Terminologies
 Antibody: A protein found in the blood that is produced in response to foreign substances
(e.g. bacteria or viruses) invading the body. Antibodies protect the body from disease by
binding to these organisms and destroying them.
 Antigens: Foreign substances (e.g. bacteria or viruses) in the body that are capable of
causing disease. The presence of antigens in the body triggers an immune response.
 Antitoxin: A solution of antibodies against a toxin. Antitoxin can be derived from either
human (e.g., tetanus immune globulin) or animal (usually equine) sources (e.g., diphtheria
and botulism antitoxin). Antitoxins are used to confer passive immunity and for treatment.
 Active immunity: The production of antibodies against a specific disease by the immune
system. Active immunity can be acquired in two ways, either by contracting the disease or
through vaccination. Active immunity is usually permanent, meaning an individual is
protected from the disease for the duration of their lives.
 Passive immunity: Protection against disease through antibodies produced by another
human being or animal. Passive immunity is effective, but protection is generally limited
and diminishes over time (usually a few weeks or months).

Mechanism of vaccine inside body

Types of vaccines
1. Live attenuated Vaccines
 Live attenuated vaccines contain whole bacteria or viruses which have
been “weakened”(attenuated) so that they create a protective immune
response but do not cause disease in healthy people.
 For most modern vaccines this “weakening” is achieved through genetic
modification of the pathogens.
 E.g. BCG vaccine, MMR vaccine, chickenpox vaccine and ect.

2. Inactivated Vaccines
 Inactivated vaccines contain whole bacteria or viruses which have been killed
or have been altered, so that they cannot replicate.
 Because inactivated vaccines do not contain any live bacteria or viruses, they
cannot cause the diseases against which they protect, even in people with
severely weakened immune systems.
 However, inactivated vaccines do not always create such a strong or long-
lasting immune response as live attenuated vaccines.
• E.g. Rabies vaccine, Japanese encephalitis vaccine, Inactivated polio virus
(IPV) and ect.

3. Subunit Vaccines
 Subunit vaccines which do not contain any whole bacteria or viruses at
all. Instead, these vaccines typically contain one or more specific antigens (or
“flags”) from the surface of the pathogen.
 The advantage of subunit vaccines over whole pathogen vaccines is that the
immune response can focus on a small number of antigen targets (“flags”).
 They usually require repeated doses initially and subsequent booster doses in
subsequent years.
 AJUVENTS are often added to subunit vaccines. These are substances which
help to strengthen and lengthen the immune response to the vaccine.

Different types of adjuvants
1. ALUM
 Most commonly used adjuvant.
 Consists of aluminum salts that
are not soluble in water.
 Used in hepatitis B vaccine.
2. VIROSOMES
 Resemble as virus, but not
infectious.
 they contain antigens and surface
protein, but not have genetic
material.
 Used for flu vaccine and hepatitis A.

4. Recombinant Protein Vaccines
• Recombinant vaccines are made using bacterial or yeast cells to
manufacture the vaccine. A small piece of DNA is taken from the virus or
bacterium against which we want to protect and inserted into the
manufacturing cells.
• For example, to make the hepatitis B vaccine, part of the DNA from the
hepatitis B virus is inserted into the DNA of yeast cells.
• These yeast cells are then able to produce one of the surface proteins from
the hepatitis B virus, and this is purified and used as the active ingredient
in the vaccine.
• E.g. Hepatitis B vaccine, HPV (Human Papilloma virus Vaccine), and etc..

5. Toxoid Vaccines
 Some bacteria release toxins (poisonous proteins) when they attack the
body, and it is the toxins rather than the bacteria itself that we want to be
protected against.
 The immune system recognizes these toxins and are able to mount an
immune response to them.
 Some vaccines are made with inactivated versions of these toxins. They are
called ‘toxoids’ because they look like toxins but are not poisonous. They
trigger a strong immune response.
 E.g. Diphtheria vaccine, Tetanus vaccine, and Pertussis (whooping cough)
vaccine.

6. RNA vaccines
 RNA vaccines use mRNA (messenger RNA) inside a lipid membrane. This
lipid membrane protects the mRNA when it first enters the body, and also
helps it to get inside cells by fusing with the cell membrane.
 Once the mRNA is inside the cell, machinery inside the cell translates it into
the antigen protein.
 This mRNA typically stimulate an immune response. It is then naturally
broken down and removed by the body.
 RNA vaccines are not capable of combining with the human genetic code
(DNA).
 E.g. COVID-19 vaccine (Moderna and Pfizer)

Antigen (foreign substance)
Antigen uptake pathway
Endogenous pathway Exogenous pathway
Antigen introduce into
body
Endogenous antigens Exogenous antigens
(Cytosolic pathway) (Endocytic pathway)
Antigen trigger’s
immune system
Antibody release
Antigen-antibody
interaction
Antigen destruction
OVERVIEW OF ANTIGEN UPTAKE MECHANISM

ANTIGEN UPTAKE PATHWAYS
There are mainly 2 types of antigen uptake pathways, endogenous and
exogenous antigen uptake pathways.
1. Endogenous Antigens- Endogenous antigens are derived from proteins
produced inside the cell.
 These includes altered self-protein antigens (e.g. tumour antigens) and non-
self protein antigens (e.g. viral antigens).
 Endogenous antigens associate with Class I MHC molecules that activate
cytotoxic CD8+ T cells for killing infected cells and tumour cells.
 Endogenous antigens can be processed and presented by any nucleated cell.

2. Exogenous Antigens-
 Exogenous antigens are derived from proteins produced outside the cell.
 These includes various bacterial, viral, protozoal, fungal and parasitic antigens which are
derived from outside the body.
 Exogenous antigens associate with Class II MHC molecules that activate helper CD4+ T cells
for providing help to B and Tc cells.
 Exogenous antigens are processed and presented by APCs (Antigen Presenting Cells).
MHC (Major Histocompatibility Complex) Molecule- They are cell surface proteins,
essential for recognizing the foreign substance and also help to get acquired
immune system. There main function is to bind with antigens of pathogens and
expose them on cell surface, in order to get them killed by T cells.

Endogenous Antigens Processing Pathway
(Cytosolic Pathway/MHC class I pathway)
The whole process is divided into 5 main
steps-
1. Processing of antigens into peptides.
2. Transfer of peptides by TAP proteins.
3. Generation of Class I MHC
molecules.
4. Association of Peptides with MHC
molecule.
5. Transport of pMHC to cell surface
and presentation.

Step I- Processing of Antigens into Peptides.
 Step-IA: Ubiquitination:
• Step-IB: Proteasome-mediated processing:

Step-II: Transfer of peptides by TAP proteins
 TAP proteins (Transporters associated
with Antigen Processing) proteins.
 TAP 1 and TAP 2 form heterodimer in
membrane of ER to facilitate selective
transport of peptides from cytoplasm
into lumen of ER.

Step-III: Generation of Class I MHC Molecule
 Calnexin is a chaperone protein that binds to newly synthesized a-chain of
Class I MHC and retains the Class I MHC from being degraded until β2-
microglobulin binds.
 Tapasin and Calreticulin both bind to the newly formed Class I MHC
complexes. Tapasin forms a bridge between the TAP proteins with the Class I
MHC molecules, whereas calreticulin prevents lodging of any other peptide
in agerotope.

Step-IV: Association of Peptides with MHC Molecule
 Peptides replaces tapasin and calreticulin and bind to the agerotope of Class I
MHC molecules to form pMHC.
 Peptide binding provides stability for Class I MHC to allow transfer to surface.
 Presentation of processed peptides in association with MHC molecules is called
pMHC.

Step- V: Transport of pMHC to cell surface
and presentation
 The pMHC-I complex is transported from ER via
Golgi bodies in a membrane bound vesicle to the
cell surface.
 The membrane of transport vesicle fuse with the
cell membrane and pMHC complex bind to
membrane toward exterior to be recognized by
CD8+ T cell.

Exogenous Antigens Processing Pathway
(Endocytic Pathway/MHC class II pathway)

ANTIGEN-ANTIBODY INTERACTION
 Antigen’s after getting recognized by CD8+ & CD4+ T cells respectively, trigger’s the immune
system of the body.
 This initiates the production and release of antibodies against the antigens from B cells along
with other immune cells.
 Which leads to antigen- antibody interaction and finally causing the destruction of antigen.

SINGLE SHOT VACCINES
Definition- single shot vaccine is a combination product of a prime component
antigen with an adjuvant and a microsphere component which encapsulates the
antigen, which will provide the booster immunization by delayed/controlled
release of the antigen.
 It is given at a single contact point for preventing 4-6 disease.
 They can replace the separate booster dose of vaccine.
 E.g. COVID-19 vaccine by Johnson & Johnson company, Single-shot Subunit Vaccine for HIV

ROLE OF MICROSPHERE COMPONENT IN
SINGLE SHOT VACCINE
 Encapsulating the booster vaccine into polymer particles should enable the
continued release of the booster dose, that when combined with a free
priming vaccine, can mimic a prime-boost regimen within a single
immunization.
 Vaccine encapsulation helps by improving immune responses through
antigen shielding, controlled antigen release and adjuvanting effect due to the
particulate nature of the encapsulated vaccine delivery system.
ADVANTAGE OF VACCINE ENCAPSULATION-
 REDUCE costs and the pressure on resources to deliver the booster doses.
 INCREASE vaccination compliance and coverage globally.
 Encapsulated antigens are protected from enzymatic degradation or rapid
elimination in vivo, further contributing to an enhanced immune response.

VACCINE ADJUVANTS
 DEFINATION- Adjuvants are the substance added to vaccine, which enhance
the working efficiency of vaccine. Presence of adjuvant, triggers the immune
response and makes them more sensitive towards the vaccine.
 NEED OF ADJUVANTS-
1. Increase the therapeutic efficacy of vaccine.
2. They form depot of antigen at site of inoculation with slow release of antigen.
3. Improve the performance of vaccine by targeting the antigen to APC.
4. Reduce the number of immunizations, required for protective immunity.

ADJUVANTS ACCORDING TO MECHANISM OF
ACTION
particulate vaccine-delivery systems Immunostimulatory adjuvants
They target antigen to antigen
presenting cells (APCs).
E.g. Liposomes, biodegradable
microspheres.
They directly activate such cells through
specific receptors e.g. toll-like
receptors (TLRs) resulting in
inflammatory responses that amplify the
innate immune response.
Activate the innate immune system
to respond more rapidly to infection
and for the adaptive immune
response to become more specific.

BIODEGRADABLE POLYMERS USED
AS VACCINE ADJUVANTS
 Biodegradable polymer of choice is the FDA-approved poly lactic-co-
glycolic acid (PLGA).
WHY PLGA??
 They can be adapted in size and/or structure to resemble a pathogen.
 To achieve the targeted delivery to promote humoral and CMI responses.
 For controlled release of antigens from polymer matrix.

MANUFACTURING OF SINGLE SHOT
VACCINE
 This single shot vaccine are prepared by microsphere encapsulation techniques.
 The most common method being “water-in-oil-in-water (W/O/W) double emulsion
solvent evaporation”.
 E.g. SINGLE DOSE TETANUS TOXOID VACCINE
 This was prepared to reduce neonatal tetanus.
 Antigen involve- PLGA microspheres encapsulating TT/ capsular polysaccharide of
Haemophilus influenzae type b (Hib) conjugated to TT.
 Method of preparation- Double emulsion water-in-oil-in-water method
 Route of administration- Directly administered into pregnant women's.

Water-in-oil-in-water (W/O/W) double emulsion solvent
evaporation method
Vaccine antigen + aq. solution
+ stabilizer (gelatin, sorbitol)
20% PLGA + methylene chloride
Water-in-oil emulsion
(W/O) emulsion
Homogenize (15000 rev/min, 30 secs)
Homogenize with 1% poly(vinyl)alcohol
W/O/W double emulsion
Solvent evaporation
Microsphere
formation
Microsphere were collected by
centrifugation followed by
washing and freeze dried.

 W/O/W emulsification method has a number of limitations that have
impeded the successful commercial development of an encapsulated antigen
in polymer microparticles for single-dose vaccines.
 Limitations like issues of antigen stability, encapsulation efficiency, particle
size and distribution and the suitable release profiles, including an initial
burst (release of antigen upon injection).
Antigen stability may be impaired due to mechanical and
chemical stress induced during emulsification steps.

W/O/W method can be adapted to produce particles of desired
size, however, it is quite difficult to achieve an extremely narrow
size distribution (monodispersity).
 The size variation is largely due to the emulsification step, during which the
particles have different droplet sizes, followed by variable precipitation rates
during the solidification phase.
 To overcome such limitations, there are certain advanced technique’s which
are carried out after double emulsification method. This technique is
commonly called as “Advanced Vaccine Encapsulation Methods”.

Advanced Vaccine Encapsulation Methods
1. Coaxial electrospraying- It is a modified version of electrospraying
technique.
 Electrospraying technique is also known as electrodynamic spraying, is
capable of producing diminutive droplets with submicron sizes by means
of an electric field. It can also be used to produce fine polymeric
particles, which are widely used for biomedical applications, particularly
drug encapsulation.
 To reduce the mechanical stress and reduce solvent-surface interactions,
one such approach is coaxial electrospraying.
 Antigen stability can also be achieved with the co-encapsulation of
stabilizing additives such as hydrophilic PEG, surfactants.

2. Encapsulation Using Microfluidics-
 Microfluidics techniques may be critical in achieving the necessary
encapsulation efficiencies for large scale vaccine manufacturing.
 Encapsulation using novel microfluidic technologies may be a promising
approach to achieve both adaptable particle sizes and a narrow size
distribution.
 Sieving the particles after emulsification can result in more uniform particle
preparations. This is referred to as extrusion and achieved with Shirasu porous
glass (SPG) beads.

MICROFLUIDICS TECHNOLOGY
 Microfluidics involves the manipulation of fluid flows at a microscale, giving the fluids a
laminar behavior.
 Microfluidics can produce highly monodispersed droplets in a controlled and repeatable
manner, a feature utilized in drug delivery, and in particular for vaccine encapsulation.
 In microfluidics, droplets are generated by intersection designs, where two immiscible or
partially miscible phases are put into contact, and subsequently produce droplets by the
combined actions of shear stress, viscous forces, and interfacial tension.
 This process can be conducted in capillaries assembled coaxially in “co-flow” type intersections
or in microfluidic chips, often made with “polydimethylsiloxane (PDMS)”.
 Polydimethylsiloxane (PDMS) is a silicon polymer poured onto a mold and hardened to form
microfluidic chips—or with glass, where the “T-junction” or “flow-focusing” designs are
embedded.
 These template multiple emulsions, either produced by capillary microfluidics or chip
microfluidics, are then converted to microcapsules.

ADVANTAGE-
• Economic
• With one injection, 4-6 infectious disease can be treated.
• Greater patient compliance
• Reduce number of doses of administration.
DISADVANTAGE-
• They can cause adverse effects.
• Act as super antigen and can over stimulate the immune response.

MUCOSAL VACCINE DELIVERY SYSTEM
INTRODUCTION-
 The adult human mucosa lines the surfaces of the digestive, respiratory,
and genitourinary tracts, covering an immense surface area (400 m2) that
is ∼200 times greater than that of the skin.
 It is estimated that 70% of infectious agents enter the host by mucosal
routes.
 The mucosal immune system consists of an integrated network of tissues,
lymphoid and nonlymphoid cells, and effector molecules such as
antibodies, chemokines, and cytokines.

 These host factors respond to pathogen invasion and infection (and to
mucosal vaccines) by triggering the innate and adaptive immune
responses to confer protection.
 Initiation of antigen-specific immune responses occurs in organized
mucosa-associated lymphoid tissue (MALT), which is characterized by the
presence of numerous lymphoid follicles covering regions of the intestinal
tract (Peyer’s patches) and respiratory tract (bronchus-associated
lymphoid tissues, BALT).
 The genitourinary tract lacks MALT, and priming of adaptive immune
responses occurs exclusively within mucosa-draining lymph nodes.

Organization of Mucosal Tissue
 Mucosal lymphoid tissues are different from peripheral lymph nodes in their anatomic
connections and orientation, cellular composition, and contribution to immune responses.
 Mucosal surfaces are typically categorized as type I or type II mucosae.
 Type I and type II mucosae are distinguished by the type of epithelium, the transport
mechanisms for immunoglobulins, the presence of organized lymphoid tissue (mucosa-
associated lymphoid tissue, known as MALT), and the composition of local immune cells.
TYPE OF MUCOSAE LOCATION
TYPE I lung and gut
TYPE II mouth, oesophagus, and cornea
BOTH (TYPE I & TYPE II) Female genital tract, type I (endocervix,
uterus) and type II (vagina, ectocervix)

SED- Sub epithelial dome
HEV- High endothelial venule

FEATURES OF TYPE I MUCOSAE
 Simple columnar epithelium linked by tight junctions covers the surfaces of type
I mucosae.
 Beneath the type I epithelial layer are organized lymphoid structures (MALT).
Microfold cells (M cells) and Dendritic cells (DCs) are present in the epithelium
covering the MALT.
 Type I mucosal epithelia express polymeric immunoglobulin receptor on their
basolateral surfaces, which binds to dimeric immunoglobulin A secreted by
plasma cells in the lamina propria.
 IgA is exported transepithelial into the lumen of type I mucosal tissue and is the
main protective immunoglobulin at these sites.
 IgA is largely protease resistant and can therefore bind and neutralize pathogens
or toxins in the gut despite the presence of active digestive enzymes.
 In both the airways and intestine, IgA provides the first barrier to invasion by
pathogens, so induction of potent IgA responses is an important goal of mucosal
vaccination.

FEATURES OF TYPE II MUCOSAE
 Stratified squamous epithelium linked by tight junctions covers the
surfaces of type II mucosae.
 Immunoglobulin G is the main protective immunoglobulin of type II
mucosal surfaces.
 Type II mucosal tissue lacks MALT at steady state, and specialized
intraepithelial DCs named Langerhans cells found in type II mucosae
within and beneath the stratified epithelium.

MECHANISM OF MUCOSAL IMMUNE SYSTEM
2. Uptake and transfer of antigen by M cells
3. Sensitization of IgA+ B cells,
CD4+Th and CD8+ CTL
4. Migration
Lymph Nodes Thoracic duct
Blood
5. Localization to effector site
6. Expansion and Maturation
• Lamina propria region
(GI, Respiratory tract)
• Glandular tissues:
(Mammary, salivary)
7. Production of IgA

MUCOSAL BARRIERS TO VACCINE DESIGN
 Direct mucosal immunization has proven difficult, due to following
challenges made by mucosal system.
 One challenge of mucosal immunization is that mucosal vaccines tend to
become diluted in mucosal fluids, and bulk flow may limit effective
deposition onto the epithelium of the mucosal system.
 Second challenge is that mucosal vaccines stuck within the mucus gel and
are subsequently degraded by proteases.
 Recent literature suggests that mucosal vaccines might be more efficient if
they were designed to mimic physicochemical properties of opportunistic
pathogens, specifically charge and size.

A variety of strategies exist for the delivery and presentation of
immunomodulatory molecules to the host immune system.
Of these strategies, those that will be effective for mucosal
immunization will require,
(a) overcoming physiological barriers at mucosal routes,
(b) targeting of mucosal APCs for appropriate processing of antigens that
lead to specific T and B cell activation, and
(c) controlling the kinetics of antigen and adjuvant presentation in order
to promote long-lived, protective adaptive immune memory
responses.

Mucoadhesion and Mucus Penetration
 Mucus is a highly viscous and heterogeneous microenvironment that
presents a significant barrier not only to pathogen entry but also to
mucosal vaccine delivery.
 mucosal vaccines must prevent inactivation of the antigen or adjuvant by
the harsh mucosal environment and deliver the vaccine across mucosal
barriers to target mucosal tissues and cells.
 Both the viscosity and pore size of mucus can block significantly the
diffusivity of agents delivered to mucosal surfaces.
 The viscosity of mucus is typically 100–10,000 times greater than the
viscosity of water. The pore size of mucus was originally estimated to be
between 20 nm and 200 nm.

 Hydrophilic and net-neutral surface chemistries are thought to promote
mucus penetration (diffusivity).
 Mucoadhesion is enhanced by highly hydrophobic or positively charged
surfaces that may interact with the negatively charged mucus layer and
block the diffusion.
 Surface modification of drug delivery vehicles has proven to be a beneficial
tool to increase both mucoadhesion and mucus penetration.
 Rajapaksa et al. showed that coating the surface of poly (lactic-coglycolic)
acid (PLGA) nanoparticles with poloxamer 188 (surfactant), helped to
increase the diffusivity and uptake of these particles by mucosal surfaces.
 (PEG) is a bioinert synthetic polymer that can function to enhance mucus
penetration or mucoadhesion on the basis of its molecular weight.
 Nanocarriers grafted with long PEG chains (≥10 kDa) are more
mucoadhesive, whereas those with shorter PEG chains (≤2 kDa) have
greater mucosal diffusivity.

S.NO NAME OF POLYMER MUCOADHESIVE BIODEGRADABLE
01 Chitosan YES YES
02 Alginate YES YES
03 Gelatin NO YES
04 Hyaluronic acid YES NO
05 Carbopol YES NO
06 Polyphosphazenes NO YES
07 Polyanhydrides NO YES
08 PLGA YES YES
09 PLA YES YES
10 Poly(caprolactone)
PCL
NO YES
11 Poly (acrylates) YES YES
12 Poly (ethylene glycol) YES NO
13 Poly (ethylene oxide) YES NO
List of
Mucoadhesive/
Biodegradable
Polymers

DESIGN STRATEGIES FOR MUCOSAL
VACCINES
Nonviral, Polymer-Based
Carrier Systems
Viral, Polymer-Based
Carrier Systems
EMULSION TYPE
LIPOSOME TYPE
VIRUS-LIKE
PARTICLE TYPE
VIROSOMES
Nanoparticles

Nonviral, Polymer-Based Carrier Systems
A. EMULSION TYPE- Nano emulsion technology
I. Water-in-oil emulsion
a. Vaccine characteristics- Th1-stimulating antigens
b. Advantages- Slow release of antigen
c. Disadvantages- Reactogenicity
II. Oil-in-water emulsion
a. Vaccine characteristics- Th2-stimulating antigens
b. Advantages- Slow release of antigen
c. Disadvantages- Reactogenicity

Nano emulsion droplets are formed by the dispersion of two
immiscible liquids.
They range in size from 20 nm to 200 nm, which is similar to the size
of opportunistic pathogens, and are readily taken up by mucosal M
cells and subsequently presented to APCs.
water-in-oil emulsions incorporate and deliver hydrophilic drugs much
more efficiently than do oil-in-water emulsions, which are used to
incorporate and deliver hydrophobic drugs.
Single-nano emulsion technology has been successfully employed in
the generation of a hepatitis B vaccine.
Unfortunately, single-nano emulsion methods have poor controlled-
release profiles.

Hanson et al. introduced the concept of a double-emulsion
method with good controlled release profiles.
Double-emulsion technology has proven useful for delivering
vaccines to mucosal surfaces before they are degraded.
 Double emulsions are more stable and are able to encapsulate
antigens without deleterious effects to the antigen during the
emulsification process.
Nano emulsion technology has provided a novel delivery method
for immunizing the mucosal immune system.

B. LIPOSOME TYPE-
a. Vaccine characteristics-Water-insoluble drugs, Water-soluble
drugs, Proteins, DNA.
b. Advantages- Easy surface modification, Synthesized from nontoxic
material, Dual function, Wide range of antigen encapsulation.
c. Disadvantages- Low antigen loading, and Low stability.
I. pH-sensitive liposomes-
a. Vaccine characteristics- DNA cytotoxic agents and Proteins
b. Advantages- Efficient endocytic release
c. Disadvantages- Intramembrane repulsion
II. Cationic liposomes-
a. Vaccine characteristics- DNA and siRNA
b. Advantages- Controlled release of antigen
c. Disadvantages- Nonspecific interactions

 Liposomes are nonpolymeric carriers that have been used extensively for
drug delivery and show great promise as vaccine carriers for mucosal
immunization.
 composed of a variety of phospholipid molecules that are based on the
structure of natural biological membrane lipids.
 Liposomes are poorly water soluble and self-assemble into a phospholipid
bilayer that can form a multilamellar or uni-lamellar vesicle that encloses an
aqueous compartment.
 The hydrophobic bilayer and the aqueous core of the liposome are suitable
for delivering lipophilic or hydrophilic cargo, respectively.

 Methods to prepare liposomes can generate small (<50-nm) or giant (>1-μm) unilamellar
vesicles.
 Cationic lipids can be tailored for complexation and efficient delivery of nucleic acids,
 pH-sensitive lipids can be tailored for pH-triggered release of agents.
 liposome-based mucosal vaccines have been used primarily for oral or intranasal
immunization.
 Rosada et al. showed that a single intranasal immunization with cationic liposomes
delivering a DNA encoding for a tuberculosis heat-shock protein could protect against
challenge with the bacterium by eliciting strong cellular immune responses.
 These stabilized liposome vaccines enhanced humoral and cellular immune responses by
10–1,000-fold.

C. Nanoparticles-
 Types of nanoparticles such as micelles, dendrimers, and solid matrix
nanoparticles composed of synthetic or natural polymers.
 Common polymer compositions of nanoparticles include biodegradable
synthetic polymers [e.g., polyesters, polyanhydrides, poly(amino acids)].
 Natural polymers (chitosan, alginate, albumin), copolymers, and polymer
blends.
 A primary goal of nanoparticle mucosal vaccine design is to protect the
antigen from degradation upon mucosal delivery, penetrate mucosal
barriers, and control the release of the antigen.

 Size plays a critical role in the amount of antigen that can be delivered as
well as the manner in which the antigen is internalized and processed by
the mucosal immune system.
 Polymeric micelles and dendrimers can be synthesized in the ultrasmall
(<25nm) size range.
 Polymeric micelles are composed of amphiphilic block copolymers that
have hydrophilic and hydrophobic segments, exhibit large solubility
differences.
 solubility difference are useful for delivering agents encapsulated within
the core or attached to the polymer shell.
 Polymeric micelles, are unstable upon dilution.
 Therefore, dendrimers can be used for noncovalent encapsulation of
vaccines or formation of covalent dendrimer-vaccine conjugates.

Dendrimers offer greater stability compared with polymeric micelles
owing to the covalent bonds that form the branched polymer
network.
polymeric micelles and dendrimers, biodegradable systems
synthesized from natural or synthetic polymers typically range in
size from 100 nm to >1 μm.
The hydrophobicity or hydrophilicity, as well as charge of the
nanoparticle surface can alter the microstructure of mucus and lead
to mucoadhesion or mucus penetration.

TECHNIQUES OF NANOPARTICLE PREPARATION
 Emulsification technique are used commonly to fabricate nanoparticles.
 Nanoparticles can be formed from emulsions through a variety of techniques
that induce polymer precipitation upon solvent removal by extraction,
evaporation, diffusion, or de-salting.
 Nanoparticles can also be fabricated by gelation of polymers dispersed in
emulsion droplets.
 This method is applicable only to polymers that exhibit gelling properties in
response to temperature, pH, or addition of cross-linking agents.

 Dispersion of monomers within emulsion droplets can also give rise to
nanoparticles by in situ polymerization.
 Particulate carriers fabricated using the copolymer PLGA represents one of
the most widely utilized delivery platforms for vaccines.
 PLGA has an excellent controlled-release profile, excellent toxicological
profiles, and US Food and Drug Administration approval.
 The surfaces of PLGA nanoparticles are easily modified, that enhance particle
diffusivity through the mucosa and transcytosis by mucosal M cells.
 One of the most important attributes of PLGA is that, vaccine antigens can be
encapsulated into the matrix or on the surface of PLGA carriers.

D. VIRUS-LIKE PARTICLE TYPE AND VIROSOMES-
• Virus-like particles (VLPs) and virosomes constitute a category of subunit vaccines wherein the
immunogens are derived from viral components that self-assemble into higher-order 3D structure
that preserve the antigenic structure of virus immunogens.
• VLPs are formed from the self-assembly of one or more viral capsid or envelope proteins that
are expressed recombinantly in mammalian or insect cells.
• Hepatitis B vaccine was the first commercially viable VLP-based vaccine.
• The human papillomavirus (HPV) vaccine has been the only other VLP since to be licensed for
human use.
• Virosomes can be regarded as a special category of liposome vaccine delivery systems
whereby viral membrane proteins are integrated into unilamellar vesicles composed of
viral and other natural or synthetic lipids.

 The most advanced virosomal systems are based on lipids (derived from
viral, egg, or synthetic lipids) and membrane proteins of influenza virus; they
are referred to as “Immunopotentiating reconstituted influenza
virosomes (IRIVs)”.
a. Vaccine characteristics- Plasmid DNA, Proteins, Peptides.
b. Advantages- Lacks viral genes, Highly immunogenic, High rate of uptake,
Undergoes self-assembly.
c. Disadvantages- Formulated by recombinant technology

TRANSDERMAL VACCINE DELIVERY SYSTEM
INTRODUCTION-
 The skin is the largest and most accessible organ of the body.
 The development of needle free immunization methods has thus become an important
goal in global health care.
 Dermal vaccination or transcutaneous immunization is a needle free method of vaccine
delivery.
 Transcutaneous immunization (TCI) via needle-free and non-invasive
drug delivery systems is a promising approach for overcoming the current
limitations of conventional parenteral vaccination methods.

ADVANTAGES-
 Ease of administration
 Reduced spread of blood-borne diseases
 Potential for generation of both systemic and mucosal immune
response.
 Assist in the implementation of multiple boosting and
multivalent vaccine regimes.
 Greater patient compliance.

SKIN BARRIER’S
Physical barrier Enzymatic barrier Immunological barrier

1. Physical barrier
• The outermost layer, the stratum corneum.
• It consists of a brick wall like structure of corneocytes in a
matrix of intercellular lipids, with desmosomes acting as
molecular rivets between the corneocytes.
• The stratum corneum presents an effective physical barrier to
the permeation of large molecules such as vaccines.
• This is the first barrier property that must be overcome to
provide effective transdermal vaccine delivery.

2. Enzymatic barrier
 The skin possesses many enzymes capable of hydrolyzing peptides and
proteins.
 These are involved in the keratinocyte maturation and desquamation
process, formation of natural moisturizing factor (NMF) and general
homeostasis.
 Their potential to degrade topically applied vaccine antigens should be
considered.

3. Immunological barrier
When the skin is damaged, environmental contaminants can access
the epidermis to initiate an immunological response, This includes
(i) Epithelial defense as characterized by antimicrobial peptides (AMP)
produced by keratinocytes.
(ii) Innate-inflammatory immunity, involving expression of pro-
inflammatory, cytokines and interferons.
(iii) Adaptive immunity based on antigen presenting cells, such as
epidermal Langerhans and dendritic cells, mediating T-cell
responses. in the lymph nodes. This promotes the generation of
both systemic (IgG and IgM) and mucosal (IgA) humoral immune
responses.

DESIGN STRATEGIES FOR TRANSDERMAL
VACCINES

A. Liquid-jet injection (Needle-free injection
devices)
 Liquid jet injectors use a high-velocity jet (typically 100 to 200 m/s) to
deliver molecules through the skin into the subcutaneous or intramuscular
region. Liquid-jet injection
multi-use nozzle jet injectors
(MUNJIs)
disposable cartridge jet injectors
(DCJIs)
depending on the number of injections carried out
with a single device.

COMPONENTS OF LIQUID JET INJECTORS
 power source (compressed gas or spring)
 Piston
 drug or vaccine-loaded compartment
 application nozzle, with orifice size in the range of
150 to 300 μm.

WORKING OF LIQUID JET INJECTOR
Upon actuation the power source
Pushes piston rapidly
increases the pressure within the drug-
loaded compartment
forcing the drug solution through the orifice
as a high velocity liquid jet
jet impacts on the skin it creates
a hole through allowing the
liquid to enter the skin.
deposited liquid can then disperse within
the tissues to illicit an immune response.

Applications of liquid-jet injectors have been focused on delivery
of macromolecules that do not passively permeate the skin.
 ADVANTAGE-
1. increase immune responses
2. reduced "dose-sparing" amounts of vaccine
3. increasing the speed
4. avoiding the risks and discomfort.
 DISADVANTAGE-
1. pain and bruising, at the site of administration.

Commercially available devices
BRAND NAME AND COMPANY NAME TYPE OF DELIVERY
Antares Vision® and Choice® (Antares,
Minneapolis)
variable dose of insulin
V-Go Mini-Ject system (Valeritas, Parsippany, NJ) variable dose of insulin
Biojector 2000 (Bioject, Tualatin, OR) for smallpox vaccination
PenJet (PenJet Corp., Santa Monica, CA) for smallpox vaccination
Injex (HNS International, Anaheim, CA) For insulin
Zeneo (Crossject, Paris, France) For human growth hormone

B. Epidermal powder immunization
 Powder injectors were first used for DNA and RNA transfection into
plants.
 The technique has subsequently been investigated for transdermal protein
delivery, gene therapy and vaccination.
 COMPONENTS- The device design principles are similar to liquid
injectors.
1. powder compartment
2. compressed carrier gas, such as helium.

Working Of Epidermal powder immunization
Actuation particles are carried by the gas
Impact the skin surface at
high velocity
puncturing micron-sized
holes in the epidermis to
facilitate skin deposition.
Immune response

 A commercial example is the Particle Mediated Epidermal Delivery
(PMED®) technology, initially developed at Oxford University, U.K. and
currently owned by Pfizer.
 PMED delivers DNA vaccines into the skin in a dry powder formulation of
microscopic gold particles and is currently in development for a range of
vaccines.
 Powder injectors offer advantages over liquids in terms of formulation and
stability issues.

C. Topical application
 A number of other methods have been investigated that can be applied to the skin, to reduce
the stratum corneum barrier, and/or carry vaccine into the skin.
Topical application
non-invasive formulation
based approaches
energy based approaches stratum corneum ablation
colloidal carriers
sonophoresis, and
electroporation
Microneedles
Thermal ablation

Colloidal carriers
1. Nanoparticles and nanocarriers-
 Nanoparticles and microparticles are polymeric particles in the nanometer
and micro meter size range respectively.
 Compounds can be incorporated into the particles in form of a solid
dispersion or a solid solution, or bound to the particle surface by physical
adsorption and chemical binding, thus allowing the particles to act as carriers
or as adjuvants for the vaccine.
 Nanoparticles administered to the skin do not permeate the intact stratum
corneum, but may accumulate in hair follicles.

2. Liposomes and elastic vesicles-
 Liposomes consist of multiple bilayers of phospholipids capable of
solubilizing both lipophilic and hydrophilic compounds within their
structure.
 Liposomes could act as skin permeation carriers, by alteration of the
composition including incorporation of surfactants, provides elastic or
deformable liposomes, claimed to be capable of deforming in shape so as to
“squeeze through” narrow pores in the stratum corneum.

Energy based approaches
 Exposure of the skin to energy in the form of electrical pulses or ultrasonic
waves can disrupt the stratum corneum barrier to increase permeability.
1. Electroporation-
 Electroporation involves the administration of electrical pulses to create
transient pores in the skin and thus increase the skin permeability to drugs
and macromolecules.
 Inovio Biomedical Corporation (Blue Bell, PA) has developed a series of
hand-held, cordless electroporation devices that have been used in vaccine
delivery studies.
Delivery of DNA vaccines into muscle or skin tissue with
electroporation systems generated robust immune responses in a
number of disease models including influenza (H5N1 and H1N1)),
human papillomavirus, and HIV

2. sonophoresis-
 Low frequency sonophoresis involves application of ultrasound waves at
frequencies between 20 to 100 kHz to the skin surface to reduce the
stratum corneum barrier and thereby increase skin permeability.
 A commercial ultrasound device, SonoPrep, for administration of local
anesthetic, was launched in 2004 but withdrawn in 2007.

3. Thermal ablation or microporation-
 Thermal ablation generates micron-size holes in the stratum corneum by
selectively heating small areas of the skin surface to hundreds of degrees.
 The heat is applied for micro- to milliseconds so that heat transfer to the
viable tissues is avoided, thus minimizing pain and damage.
 Using this technique, a 100-fold increase in reported gene expression was
obtained following application to mice of an adenovirus vaccine carrying a
melanoma antigen.
 Commercially available examples are the PassPort® system by Altea
Therapeutics Corp (Altanta, GA) and the ViaDerm® device by TransPharma
Ltd (Israel).

4. Microneedles-
 Microneedles consist of pointed micro-sized projections, fabricated into arrays with
up to a hundred needles, that penetrate through the stratum corneum to create
microscopic holes, thus providing delivery pathways for vaccines and drugs.
 Different microneedle systems have been investigated including:
1. solid microneedles that pierce the skin to increase permeability allowing the
vaccine solution to then be applied via the skin surface.
2. solid microneedles coated with dry powder vaccine for dissolution in the skin.
3. microneedles composed of polymer with encapsulated vaccine for rapid or
controlled release in the skin.
4. hollow microneedles through which the vaccine solution can be infused into the
skin.

a. solid microneedles for permeabilizing
skin via formation of micron-sized holes
b. solid microneedles coated with dry
drug or vaccine
c. polymeric microneedles with
encapsulated drug or vaccine
d. hollow microneedles

1. Solid or insoluble microneedles-
 They are generally composed of metal such as titanium or silicone.
 The microneedles permeabilize the skin by forming micron-sized holes though the stratum
corneum.
 The microneedle array is then removed and a drug/vaccine containing patch is applied.
 This approach is termed “poke & patch”.

2. Coated microneedles-
 have an insoluble core coated with drug
that dissolves off within the skin.
 This approach is termed as “coat & poke”
approach.
3. Polymer microneedles-
• It contain the drug or vaccine in a
solid solution of needle that
dissolves, swells or degrades on
skin insertion, then releasing the
drug or vaccine.

4. hollow microneedles-
 Insoluble hollow microneedles create holes through which the drug solution can pass
into the skin.
 This approach is called as “poke & flow” approach.
• The development of insoluble solid and hollow microneedles is most
advanced for vaccine delivery.
• Advantage- No significant adverse effects from microneedles,
including minimal pain.

• Some examples of microneedle technologies-
1. ChimeriVaxTM-JE for yellow fever tested in primates.
2. Plasmid DNA encoding hepatitis B surface antigen.
3. Recombinant protective antigen of Bacillus anthracis for anthrax tested in
rabbits.
4. “4pox DNA vaccine” was administered by skin electroporation using
plasmid DNA-coated microneedle arrays. This was the first vaccine study in
which microneedle-mediated electroporation has been used to immunize
animals.

REFERENCES:
 Review on Technological Approaches for Improving Vaccination Compliance and Coverage,
Vaccines 2020, 8, 304; doi:10.3390/vaccines8020304.
 Transdermal delivery of vaccines by Sarika Namjoshi and Heather A.E.
 Mucosal Delivery of Vaccines: Role of Mucoadhesive/Biodegradable Polymers.
 Electrospraying: Possibilities and Challenges of Engineering Carriers for Biomedical
Applications—A Mini Review
 Coaxial electrospraying of biopolymers as a strategy to improve protection of bioactive food
ingredients.
 Biodegradable Polymer Microspheres as Vaccine Adjuvants and Delivery Systems by R. K.
Gupta, A. -co Chang, G. R. Siber.
 Antigen processing and presentation by rakesh sharda department of veterinary microbiology.
 https://www.slideshare.net/JanuVashi/vaccine-delivery-system.
 https://www.slideshare.net/ManishJajodiya/vaccins-drug-delivery-system.
 https://www.slideshare.net/NaveenBalaji32/single-shot-vaccines-naveen-Balaji.

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