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Dept. Of Pharmaceutics, JSSCP, Mysore 7
4. REVIEW OF LITERATURE
4.1.Vaccination
Vaccination as a deliberate attempt to protect human beings against disease
has a long history and more widespread use of vaccines could prevent 1.6 million
deaths a year, among children less than five years of age. Over the next few years a
new generation of vaccines will become available that could save the lives of up to 10
million individuals: e.g. vaccines against diarrhoeal diseases, hepatitis C, malaria,
acquired immunodeficiency syndrome (AIDS), sexually transmitted and other
diseases8. Current development efforts seek combination vaccines that protect against
multiple pathogens, in keeping with the ultimate goal of combining all the antigens
recommended for routine immunization into a single multivalent product 9.
Immunization has been the most important way of combating viral and
bacterial infections. Traditional vaccines are mainly composed of heat-inactivated
bacteria or virus. This vaccine concept has been proved effective in controlling and
even eradication of many infectious diseases.
Still newer approaches to vaccine development are urgently needed for two
reasons. First there are several infectious diseases for which the traditional approaches
have failed and for which vaccines still needed to be developed as shown in Table 1
and second, the regulatory authorities now require vaccines to meet extremely high
standards of safety and chemical-physical characterization.
Newer trends for vaccine designing include sequencing of bacterial genome
using genome technology followed by proteomics, which involves identification of
whole sets of proteins encoded by an organism.
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Dept. Of Pharmaceutics, JSSCP, Mysore 8
In-vivo expression technology (IVET) and signature tagged mutagenesis
(STM) are the two new methods that have been formulated to isolate genes that are
specifically induced during infection.
These above mentioned modern techniques have given rise to effacious
vaccines like hepatitis B vaccine, Bordetella pertusis vaccine and vaccine against
Helicobacter pylori Serogroup B by reverse vaccinology 10.
However, the success of immunization is not only dependent on nature of
immunogenic components, but also on the preparation form. Newly developed
vaccine formulations have to satisfy detailed criteria to guarantee the highest possible
quality, safety and efficacy standards.
The majority of pathogens invade the body via one or more of the mucosal
routes. Oral, nasal, pulmonary, and urino-genital routes are the most common
pathways for entry of infectious pathogens into the human host. Therefore, the
importance of generating a �first-line of defense� at the site of entry has been well
recognized. In order to have adequate mucosal protection, there are several factors
that can influence the effectiveness of vaccines and amongst them, the most critical
factor in mucosal vaccine effectiveness is the route of administration and potential for
the antigen to be processed by the antigen-presenting immune cells, such as
macrophages and dendritic cells. Presently, most vaccines are administered via the
parenteral route or via other invasive routes. It is critically important to examine the
development of mucosal vaccination strategies that can effectively trigger systemic as
well as mucosal immunity.
Polymeric nanoparticles are also actively being investigated for vaccine
delivery. For instance, Estevan and coworkers have encapsulated extracts of
Salmonella enterica serovar Abortusovis in polymeric nanoparticles 11. Upon
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Dept. Of Pharmaceutics, JSSCP, Mysore 9
subcutaneous injection in Balb/c mice, the nanoparticles conferred a significant
protection and the antibody titer levels were similar to those induced by the attenuated
commercial vaccine Rv6. In another study, ovalbumin was encapsulated in alginate-
coated chitosan nanoparticles and delivered to Peyer�s patches 12.
4.2. Nanotechnology
Nanotechnology refers broadly to a field of applied science and technology
whose unifying theme is the control of matter on the molecular level in scales smaller
than 1 µm, normally in size ranges from 1-100 nm, and the fabrication of particles
within that size range. Nanotechnology is expected to bring a fundamental change in
manufacturing in the next few years and will have an enormous impact on life
sciences including drug delivery, diagnostics, nutraceuticals and production of
biomaterials. Engineered nanoparticles (NP) (<100nm) are an important tool to realize
a number of these applications. Conceptually nanoparticle is a collective name for
both nanospheres and nanocapsules. Drugs may be absorbed at the sphere surface or
encapsulated within the particle. Nanoparticles now days are receiving considerable
attention for the delivery of therapeutic drugs. Uptake of nanoparticles prepared from
hydrophobic polymers seems to be higher than that of particles with more hydrophilic
surfaces 13, thus more hydrophilic particles may be rapidly eliminated.
The potential advantages of nanoparticles as oral drug carriers are enhancement of
bioavailability, delivery of vaccine antigens to the gut-associated lymphoid tissues
(GALT), controlled release, and reduction of the gastrointestinal irritation caused by
drugs 14.
Depending on their composition and intended use, nanoparticles can be
administered orally, parenterally, or locally 15-18. A number of different strategies have
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Dept. Of Pharmaceutics, JSSCP, Mysore 10
been proposed in order to modify the physicochemical characteristics of the
nanoparticles and, thus, their interactions within a biological environment. For
example, it is possible to change the chemical nature of the polymeric matrix of the
nanoparticles and thereby alter certain biological phenomena such as biorecognition,
biodistribution, bioadhesion, biocompatibility, and biodegradation. Some polymeric
materials used for this purpose are gelatin, chitosan, sodium alginate, poly (alkyl
cyanoacrylates), poly- (lactic acid), poly(lactic-co-glycolic acid), poly[ethylene
glycol- co-(lactic-glycolic acid)], poly(-caprolactone), and poly- (methyl
methacrylate) 19-20.
It is well known that the reticuloendothelial system, mainly the liver and
spleen, is a major obstacle to active targeting because of its ability to recognize these
systems, remove them from systemic circulation, and, consequently, avoid the
effective delivery of the nanospheres to organs other than those of the
reticuloendothelial system 21. Surface characteristics of nanoparticles can be easily
manipulated to achieve both passive and active drug targeting. Site-specific targeting
can be also be achieved by attaching targeting ligands to surface of particles or use of
magnetic support.
Knowledge of the fundamental relationships would allow nanoparticles to be
designed with defined size and surface characteristics for delivery to specific cells or
organs in body. On the other hand, polymeric nanoparticles offer some specific
advantages over liposomes. For instance, they help to increase the stability of
drugs/proteins and possess useful controlled release properties 22, 23.
4.2.1. Channels of Uptake
Nanoparticles have to cross the gastrointestinal barrier either by passive
diffusion via transcellular or paracellular pathways to deliver their drug content in the
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Dept. Of Pharmaceutics, JSSCP, Mysore 11
blood, lymph, or target organs. Another possible mechanism for the transport of
nanoparticles across intestinal cells is paracellular uptake via aqueous channels. In
humans, the equivalent pore diameter has been estimated to be between 4 and 8 A°
and about 10�15 A° in rat and rabbit 24. The epithelial transport of larger molecules or
particles can be increased by reversibly increasing the permeability of the tissue by
opening the tight junctions under the influence of some mucoadhesive polymers and
penetration enhancers 25.
4.2.2. Methods of preparation
Many methods have been developed for preparing nanoparticles; these
methods can be classified into two main categories according to formulation need
whether the formulation requires a polymerization reaction or is achieved directly
from a macromolecule or preformed polymer 26.
4.2.2.1. Interfacial polycondensation
Polymeric nanoparticles can be also prepared by the interfacial
polycondensation of the lipophilic monomer, such as phtaloyldichloride and the
hydrophilic monomer, diethylenetriamine, in the presence and absence of the
surfactant 27. These nanoparticles were smaller than 500 nm.
4.2.2.2. Emulsification/solvent evaporation
Emulsification-solvent evaporation involves two steps. The first step requires
emulsification of the polymer solution into an aqueous phase. During the second step
polymer solvent is evaporated, inducing polymer precipitation as nanospheres. A
polymer organic solution containing the dissolved drug is dispersed into nanodroplets,
using a dispersing agent and high-energy homogenization 28, in a nonsolvent or
suspension medium such as chloroform (ICH, class 2) or ethyl acetate (ICH, class 3).
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Polymer get precipitated in form of nanospheres in which drug is finely dispersed in
the matrix of polymer. Further to evaporate the solvent, temperature is increased
under pressure or by continuous stirring. The size of nanospheres can be controlled by
adjusting the stir rate, type and amount of dispersing agent, viscosity of organic and
aqueous phases, and temperature.
4.2.2.3. Solvent displacement and interfacial deposition
Solvent displacement method is based on principle of spontaneous
emulsification of the organic internal phase in which polymer had been already
dissolved into the aqueous external phase. However, there is difference in products
achieved. Solvent displacement leads to formation of nanospheres or nanocapsules.
Solvent displacement involves the precipitation of a preformed polymer from an
organic solution and the diffusion of the organic solvent in the aqueous medium in the
presence or absence of a surfactant 29-32.
The solvent displacement technique allows the preparation of nanocapsules when a
small volume of nontoxic oil is incorporated in the organic phase. Considering the oil-
based central cavities of the nanocapsules, high loading efficiencies are generally
reported for lipophilic drugs when nanocapsules are prepared 33.
4.2.2.4.Emulsification/solvent diffusion
Emulsification/solvent diffusion (ESD) usually emphasizes the use of organic
solvents. In this method the encapsulating polymer is first dissolved in a partially
water soluble solvent (propylene carbonate) which is further saturated with water (to
maintain initial thermodynamic equilibrium of both liquids).
For the formation of nanoparticles, it is necessary to promote the diffusion of the
solvent of the dispersed phase by dilution with an excess of water when the organic
solvent is partly miscible with water. Subsequently, the polymer-water saturated
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Dept. Of Pharmaceutics, JSSCP, Mysore 13
solvent phase is emulsified in an aqueous solution containing stabilizer, leading to
solvent diffusion to the external phase and the formation of nanospheres or
nanocapsules, according to the oil-to-polymer ratio. Finally, the solvent is eliminated
by evaporation or filtration, depending upon its boiling point.
Several drug-loaded nanoparticles were produced by the ESD technique, including
mesotetra(hydroxyphenyl) porphyrin-loaded PLGA (p-THPP) nanoparticles34,35
doxorubicin-loaded PLGA nanoparticles36, plasmid DNA-loaded PLA
nanoparticles37, coumarin-loaded PLA nanoparticles38, indocyanine39, cyclosporine
(Cy-A)-loaded gelatin and cyclosporin (Cy-A)-loaded sodium glycolate
nanoparticles40.
4.2.2.5. Salting out method
Salting -out method is based on the separation of a water miscible solvent
from aqueous solution via a salting-out effect with the help of a salting out agent
(electrolytes, such as magnesium chloride, calcium chloride, and magnesium acetate,
or non- electrolytes such as sucrose).The salting-out procedure can be considered as a
modification of the emulsification/solvent diffusion. Polymer and drug are first
dissolved in a solvent such as acetone, which is emulsified into an aqueous gel
containing the salting-out agent and a colloidal stabilizer such as
polyvinylpyrrolidone. This oil/water emulsion is sufficiently diluted with water or
aqueous solution to enhance the diffusion of acetone into the aqueous phase, thus
inducing the formation of nanospheres.
The selection of the salting-out agent is important, because it can play an
important role in the encapsulation efficiency of the drug. Both the solvent and the
salting-out agent are then eliminated by cross-flow filtration 33.
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4.3.Applications of Nanoparticles
Table 1 enumerates various drugs which have been used in nano drug delivery
systems.
Table 1: List of drugs used in nanoparticles drug delivery
Drug Carrier System References
Antifungal drugs Submicronized emulsion 41
Camptothecin Solid lipid nanoparticles 42
B-Cyclodextrin Nanosphere 43
Danazol Lipid-based emulsion 44
Flurbiprofen Nanosuspension 45
Mitoxantrone Magnetic nanoparticles 46
UCB-35440-3 Nanocrystal 47
Cephalosporin Nanoconugates 48
Betamethasone CaCo3 nanoparticles 49
Table 2 Marketed nanomedicines developed using Nanocrystal Technology.
S No Brand Name Active Ingredient Marketed places
1 Avinza Morphine sulphate Marketed in US
2 Emend Aprepitant, poorly water soluble
compound
Marketed in US
3 Herbesser Diltiazem Marketed in Japan
4 Naprelan Naproxen sodium Marketed in US
5 Rapamume Sirolimus (immunosuppressant) Marketed in US
6 Theodur Theophyyline Marketed in Japan
7 Verelan PM Verapamil Marketed in US
8 TriCor Fenofibrate Marketed in US
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Table 3: List of Nanoparticluates medicine approved by FDA.
Brand Name Active Ingredient Developed by
Company
Indications
Triglide Nanocrstalline
fenofibrate
SkyePharma First
Horizon
Lipid disorders
Estrasorb
Estradiol
hemihydrate
micellar
Nanoparticles
Novavax Reduction of
vasomotor
symptoms
Adagen
Pegylated
adenosine
deaminase
Enzon Enzyme
replacement
therapy
Amphotec Colloidal
suspension of lipid
based amphotericin
B
Sequus For invasive
aspergillosis
pateints
Oncaspar
Pegasparginase Enzon Leukemia
Elestrin Estradiol gel
containing calcium
phosphate
nanoparticles
BioSante Treatment for
moderate to severe
hot flashes in
menopausal
womens
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Dept. Of Pharmaceutics, JSSCP, Mysore 16
Table 4: List of Nanoparticluates medicine under clinical trials by FDA 50.
Brand Name Active
Ingredient
Developed by
Company
Indications Status
Vivagel
Dendrimer gel
StarPharma
Holdings
Vaginal
microbicide for
the
preventionof
HIV and
genital herpes
Phase II
NB-00X
Nanoemuslion
droplets
Nanobio Herpes labialis
caused ny
herpes simplex
I virus
Phase II
Aurimmune
(CYT-6091)
Colloidal gold
nanoparticles
CytImmune
Sciences
Solid tumours Phase II
INGN-401 Liposomes
FUS-1
Introgen
Therapeutics
Metastatic, non
small cell lung
cancer
Phase I
SGT-53
p-53 liposomes Synergene
Therapeutics
Solid tumours Phase I
4.3.1. Nanoparticles for oral delivery of peptides and proteins
Protein drug-delivery technologies are of ever increasing importance because
of new therapeutic proteins and peptides which are being discovered. Significant
advances in biotechnological and biochemistry aspects have led to the discovery of a
large number of bioactive molecules and vaccines based on peptides and proteins.
Encapsulation of bioactive molecules in polymeric nanoparticles protects them
against enzymatic and hydrolytic degradation. For instance, it has been found that
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Dept. Of Pharmaceutics, JSSCP, Mysore 17
insulin-loaded nanoparticles have preserved insulin activity and produced blood
glucose reduction in diabetic rats for up to 14 days following the oral administration51.
There are three possible pathways for protein and peptide drug absorption
through the GI tract. The first is via the M-cells of Payer�s patches, the second via a
transcellular route involving enterocytes, and the third via paracellular avenues
through tight junctions 52, 53. The nanosystems are providing a viable alternative for
these drugs such as liposomes, polymeric micelles, and NPDDSs (Nanoaprticles drug
delivery systems). Rodrigues et al. have reported an interesting work on lectin
nanocarrier conjugate. They used dextran/poly(e-caprolactone) polyester polymers
and conjugated with three different proteins, lectins from leaves of Bauhinia
monandra and Lens culinaris, and bovine serum albumin (BSA). The nanoparticles
having a size around 200 nm could be used for delivering proteins 54.
4.3.2. Nanoparticles as a vaccine adjuvant
There are at least three reasons that nanoparticles might have an advantage
compared to microparticles. The first reason is that nanoparticles have increased
surface area for adsorption allowing for a higher antigen: polymer ratio. The second
reason is possible enhanced immunogenicity, although the evidence is conflicting. It
is well established for vaccine delivery that < 5 mm particles offer enhanced immune
responses compared to larger particles. Particle uptake by macrophages has been
found to be optimal for smaller microparticles (< 3 mm). There is evidence that even
smaller nanoparticles have increased uptake, which suggests that nanoparticles may
enhance immune responses compared to microparticles. The third reason
nanoparticles have advantage over microparticles is preparation and processing
simplifications for nanoparticles. For this to be an advantage compared to
microparticles, the exact preparation method for the nanoparticles is important.
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Dept. Of Pharmaceutics, JSSCP, Mysore 18
Microparticles are typically prepared by the double-emulsion solvent
emulsification evaporation method. These methods uses water/ oil/water double
emulsion that is formed by high shear homogenization. Nanoparticles of approximate
size 200 nm can also be created by a modified solvent emulsification evaporation
method, the same method had been used to create microparticles also 55 however this
has no advantage over microparticles. An alternate nanoparticle preparation method
that offers a wide range of particle size is the solvent displacement method and was
first described earlier 29. This technique uses a water miscible solvent to produce a
one-phase mixture by magnetic stirring. These nanoparticles, depending on the
solvent, polymer type, polymer concentration, and addition of emulsifiers, can range
in size from 50 to 500 nm 56, 57. Various alternative approaches for nanoparticle
preparation have also been described based on emulsions, including spray drying, 58
and phase separation, 59, 60 however the solvent displacement method is the simplest.
The solvent displacement method has clear advantages compared to the other method
to make microparticles because it is single phase that does not require high shear
homogenization.
Couvreur and coworkers have developed insulin loaded poly
(isobutylcyanoacrylate) nanocapsules that upon oral administration, gave results
suggesting that nanocapsules could deliver insulin directly to the blood 61 and cause a
dramatic reduction of blood glycaemia, following oral administration to diabetic rats
51. Alphandary and coworkers had shown, based on TEM observations, that the same
nanocapsules were absorbed by the intestinal epithelial cells, leading to the transport
of insulin through the intestinal mucosa. However, nanocapsules were highly
degraded upon transport across M cell containing epithelium 62.
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Peptide and protein encapsulation in polymeric nanocarriers have also been
applied to oral vaccination applications. Locally produced secretory IgA constitutes
over 80% of all antibodies produced in mucosa associated tissues 53, 64 and are
considered to be among the most important protective humoral immune factors.
Furthermore, a fascinating feature of mucosal immunology is that
administration of antigen in one mucosal site can lead to generation of immune
responses not only locally but also at distant mucosal sites, a phenomenon referred to
as common mucosal immune system. Finally, mucosal immunization has also the
potential to elicit an immune response against infectious diseases for which current
parenteral vaccines either have a low efficiency or are inexistent, such as vaccines
against HIV and tuberculosis 65, 66.
Eldridge and coworkers have asserted that microspheres smaller than 5 ìm in
diameter were transported by M cells through the efferent lymphatic macrophages,
numerous microparticulate systems were developed to reach the exiting goal of oral
immunization 67, 68. Among the vehicles developed during these last years, polymeric
biodegradable microparticles, mainly composed of PLA or PLGA, have been
probably the most extensively studied. A great number of proteins have been
successfully encapsulated in PLGA microparticles with a full maintenance of
structural and immunologic integrity 69-73.
As discussed above, particles in the nanoscale size rather than microscale size
are more adapted for cellular uptake in the GI tract. Up to now, few studies have
examined the capacity of biodegradable loaded-nanoparticles to induce an in-vivo
immune response after oral administration. Jung et al. 74 have used a poly (vinyl
alcohol)-co-PLGA to reach a high level of tetanus toxoid loading by adsorption.
Nanoparticles given per os to mice induced seric IgG and IgA immune responses, the
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IgA titers being significantly higher than the control (intra peritoneal administration).
Particle size was found to significantly affect the induction of antibody production,
smaller particles inducing higher titers. In addition, cholera toxin B subunit (CTB)
entrapped in nanoparticles (420 nm) caused comparable immunogenicity than the
potent oral adjuvant, cholera toxin 75. The influence of the dose on the serum IgG
response after oral delivery of BSA at a dosing range from 50 to 200 ìg, entrapped in
1 ìm PLGA nanoparticles has elicited a systemic IgG dose/response relationship76.
The influence of particle size on the immune response after oral delivery of
BSA entrapped in 200, 500 and 1000 nm PLGA particles have also been studied 77. In
contrast to the results found in the literature over the more extensive intestinal
absorption of smaller particles, a higher serum IgG antibody levels and a similar
IgG2a/IgG1 ratio have been observed working with 1ìm particles than with the 200
and 500 nm particles.
Development of vaccine or antigen engineered nanocarriers are expected to be
immunologically more effective over conventional dosage forms since, they can be
fabricated to specifically target and be retained at the desired site of action. Vaccines
are still in need for some of the pathogenic diseases as shown in Table 5.
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Table 5: Vaccines are needed for following pathogens.
Viral Bacterail Parasitic
HIV Mycobacterium leprae Ascaris
Cytomegalovirus Gonococcus Plasmodium
Chikenguniya Escherichia coli and
Proteus
Herpes simplex (urinary tract infection) Schistosomes
Herpes Zoster Enterotoxigenic Trypanosomes
HCV Escherichia coli Ancyclostoma
Para influenza virus Streptococcus pyogenes
Respioratory synctial virus Duodenale
Lassa virus Streptococcus mutans Nactar americanus
Filoviruses Klebsiella Trichuris
(malburg ebola) Pseudomonas Filaris
Papillomavirus Shigella Giardia
Campulobacter,
Helicobacter pylori,
Staphylococcus aureus
Leishmania
4.3.3. Ocular Applications of Nanoparticulate Drug-Delivery System
Due to cornea�s low permeability to drugs and non corneal factors (rapid tear
turnover, nasolacrimal drainage, and systemic absorption) topical ophthalmic drugs
face poor absorption in the eye. Major problem in ocular delivery is to maintain an
adequate concentration of the therapeutic agent in the precorneal area. Topical drop
administration of ophthalmic drugs in aqueous solutions results in extensive drug loss
due to tear fluid and eyelid dynamics 78-80. Polymeric nanoparticles are attractive
colloidal systems because they demonstrate increased stability and have a longer
elimination half-life in tear fluid (up to 20 min), than do conventional drugs applied
topically to the eye, which have half-lives of just one to three minutes. Nanoparticles
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drug delivery system have been evaluated for ocular applications to enhance
absorption of thera-peutic drugs, improve bioavailability, reduce systemic side effects,
and sustain intraocular drug levels 81. PLGA has been evaluated and proved to be a
very useful biodegradable polymer for nanoparticluates formulation due to its medical
use, biocompatibility, and safety 82.
4.3.4. Nanoparticulate Drug-Delivery Systems for Pulmonary Treatment
Several studies have exhibited the absorption of high-molecular-weight drugs
such as insulin, heparin, and GCSF (recombinant human granulocyte colony
stimulating factor) through pulmonary drug delivery system 83. As these peptides have
a short life, the development of delivery systems with sustained pharmacological
action would be very useful. An enormous diversity of therapeutic agents is currently
administered to the patients via aerosol inhalation, and the number of potential drug
candidates for pulmonary application increase daily. The major areas of research and
therapeutic applications are asthma 84, cystic fibrosis 85, lung cancer 86, tuberculosis 87,
pulmonary hypertension 88, and diabetes 89. Some of the polymers such as PLGA,
protamine, thiomer, and lipid-based particles can be loaded by VIP or new designed
analogs. Insulin-loaded PBCA NPs were studied by Zhang Q. 90; they demonstrated
that the pulmonary administration of these nanoparticles could significantly prolong
the hypoglycemic effect of insulin.
4.3.5. Nanoparticulate Drug-Delivery Systems for Central Nervous System
Hydrophilic substances with high molecular weight have minimal passive
permeation because BBB (blood brain barrier) favors lipid soluble molecules via
passive diffusion. Transport across BBB is additionally regulated by a number of
transporters including very effective efflux transporters such as multidrug resistance-
associated protein or p-glycoprotein. Several strategies have been tried to cross the
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BBB; one alternative strategy is to use drug-carrier systems such as liposomes,
antibodies, and nanoparticles 91. Numerous studies have shown the applications of
nanoparticles for brain targeting 92. It has been reported poly(butylcyanoacrylate)
nanoparticles was able to deliver hexapeptide dalargin, doxorubicin and other agents
into the brain which is significant because of the great difficulty for drugs to cross the
BBB 93.
4.3.6. Mucoadhesive Nanoparticulate Drug-Delivery Systems
Mucosal surfaces are the most common and convenient routes for delivering
drugs to the body. However, macromolecular drugs such as peptides and proteins are
unable to overcome the mucosal barrier and are degraded before reaching the
bloodstream. Nanoparticles drug delivery systems show a promising strategy for
delivering drugs through mucosa. Studies shows that mucosal surfaces are most
common and offers ease of delivering drugs to the body however, it was found that
macromolecular drugs such as peptides and proteins are unable to overcome the
mucosal barrier and are degraded before reaching the systemic circulation.
Polysaccharide Chitosan (CS) is mucoadhesive and CS nanoparticles, CS-coated oil
nanodroplets (nanocapsules), and CS-coated lipid NPs have shown interesting
possibilities for this purpose 94.
Nanoprecipitation techniques using PLGA and PLA polymers were found to
be useful for nanoparticulate delivery of proteins and have shown more versatility and
flexibility in the formulation for protein delivery 95.
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4.3.7. Nanoparticulate Drug Delivery in Cancer Treatment
Several nanoaprticle drug delivery system are reported for the application in
cancer therapy, transferring conjugated paclitaxel-loaded nanoparticles 96,
nanovaccines 97, adriamycin-loaded nanoparticles for hepatoma treatment 98, magnetic
PBCA nanospheres with aclacinomycin A in gastric cancer 99, near-infrared
absorption nanospheres 100, polypropylenimine dendrimer nanoparticles for
oligonucleotides 101, lytic-peptide-bound magnetite nanoparticles for breast cancer
treatment 102, ceramic-based nanoparticles entrapping water-insoluble
photosensitizing anticancer drugs 103, and poly(epsilon-caprolactone) nanoparticles for
the delivery of Tamoxifen for breast cancer treatment 104.
4.3.8. Nanoparticles for gene delivery
Polynucleotide vaccines work by delivering genes encoding relevant antigens
to host cells where they are expressed, producing the antigenic protein within the
vicinity of professional antigen presenting cells to initiate immune response. Such
vaccines produce both humoral and cell-mediated immunity because intracellular
production of protein, as opposed to extracellular deposition, stimulates both arms of
the immune system. Nanoparticles loaded with plasmid DNA could also serve as an
efficient sustained release gene delivery system due to their rapid escape from the
degradative endo-lysosomal compartment to the cytoplasmic compartment 105. Hedley
et al. 106 reported that following their intracellular uptake and endolysosomal escape,
nanoparticles could release DNA at a sustained rate resulting in sustained gene
expression. This gene delivery strategy could be applied to facilitate bone healing by
using PLGA nanoparticles containing therapeutic genes such as bone morphogenic
protein.
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4.4.Characterization of Nanoparticles
Characterization of nanoparticles is an important prerequisite before drug
delivery take place and many of the tools employed for their characterization are the
same as those used for similar analysis of other submicrometer colloids such as
micelles, liposomes, and emulsions.
4.4.1. Dynamic Light Scattering (DLS)
DLS, also known as photon correlation spectroscopy (PCS) or quasi-elastic
light scattering (QELS) records the variation in the intensity of scattered light on the
microsecond time scale 107, 108. This variation results from interference of light
scattered by individual particles under the influence of Brownian motion, and is
quantified by compilation of an autocorrelation function. This function is fit to an
exponential, or some combination or modification thereof, with the corresponding
decay constant(s) being related to the diffusion coefficient (s). Using standard
assumptions of spherical size, low concentration, and known viscosity of the
suspending medium, particle size is calculated from this coefficient.
4.4.2. Static Light Scattering/Fraunhofer Diffraction
Static light scattering (SLS) is an ensemble method in which the pattern of
light scattered from a solution of particles is collected and fit to fundamental
electromagnetic equations in which size is the primary variable 109.
Fraunhofer (light, laser) diffraction is frequently employed as a sizing method for
nanoparticles. As size drops into the submicrometer regime the differences in the
scattering pattern occur primarily at high angles, so collecting such data becomes
critical�an ability that varies widely among commercial instruments. The
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approximations implemented in Fraunhofer theory are acceptable for particles of
diameter 2 mm and higher 110.
4.4.3. Acoustic Methods
Another ensemble approach, acoustic spectroscopy, measures the attenuation
of sound waves as a means of determining size through the fitting of physically
relevant equations 111. In addition, the oscillating electric field generated by the
movement of charged particles under the influence of acoustic energy can be detected
to provide information on surface charge. This is termed electroacoustic spectroscopy
and can also be reversed so that sound waves generated by the oscillatory motion of
charged particles in a varying electric field are observable 112.
4.4.4. Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) can be used to determine both the size
and the qualitative nature of nanoparticles. The selectivity afforded by chemical shift
complements the sensitivity to molecular mobility to provide information on the
physicochemical status of components within the nanoparticle 113.
4.4.5. Single-Particle Optical Sensing (SPOS)
A particle counting method, SPOS, which is also known as optical particle
counting, involves recording the obscuration or scattering of a beam of light that
results from the passage of individual particles through a sensor 114. Particles of
diameter less than 1 mm are largely undetected, thus making SPOS very useful in the
determination of the few large particles in a population that may represent a safety
concern, indicate a problem in production, or be harbingers of instability. Drawbacks
include the possible dissolution of analyte during analysis, the large dilution required,
and the need for low backgrounds.
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4.4.6. Optical Microscopy
Most nanoparticles are below the resolution limit (ca. 0.5 mm) of direct optical
imaging, though microscopy is still useful to get an estimate of size and crystallinity
of starting materials, as might be desirable in the instance of comminution or
homogenization processing, or other larger particles 115.
4.4.7. Electron Microscopy
Scanning and transmission electron microscopy, SEM and TEM, respectively,
provide a way to directly observe nanoparticles, with the former method being better
for morphological examination 116-119. TEM has a smaller size limit of detection, is a
good validation for other methods, and affords structural information via electron
diffraction, but staining is usually required, and one must be cognizant of the
statistically small sample size and the effect that vacuum can have on the particles.
4.4.8. Atomic Force Microscopy (AFM)
In this technique, a probe tip with atomic scale sharpness is rastered across a
sample to produce a topological map based on the forces at play between the tip and
the surface. The probe can be dragged across the sample (contact mode), or allowed to
hover just above (noncontact mode), with the exact nature of the particular force
employed serving to distinguish among the subtechniques. That ultrahigh resolution is
obtainable with this approach, which along with the ability to map a sample according
to properties in addition to size, e.g., colloidal attraction or resistance to deformation,
makes AFM a valuable tool. However, size and shape has been the most common
application to date 120-121. Nanoparticles are typically presented as an evaporated
suspension on a smooth silicon or mica surface, though not without the possibility of
deformation 122.
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4.4.9. Hydrophobic Interaction Chromatography
In this method the analyte is first adsorbed onto a chromatographic stationary
phase using a high concentration of an antichaotropic salt 123. Elution occurs using a
gradient in which the salt concentration is decreased, so that those materials eluting
first are the least hydrophobic because the salt concentration did not need to be
decreased much before the analyte desorbed. Originally developed for proteins,
hydrophobic interaction chromatography has been pressed into service as a means of
characterizing the hydrophobicity of nanoparticle surfaces, a property influenced by
the choice of surfactant and/or polymer and also a key parameter in determining their
in vivo fate 124, 125.
4.4.10. Ispycnic Centrifugation
Another bioanalytical method applied to nanoparticles is centrifugation of
analyte using a sucrose gradient as the suspending media. Under the influence of
Stokes� laws, sedimenting particles will settle until they reach a point where their
density matches that of the gradient. This self-focusing separation allows nanoparticle
density to be determined, which along with particle size and bulk substituent
concentration can in turn be used to calculate a number concentration 126.
4.4.11. Zeta Potential
Zeta potential is used as a surrogate for surface change, and is often measured
by observing the oscillations in signal that result from light scattered by particles
located in an electric field 127, 128.
4.4.12. Differential Scanning Calorimetry (DSC)
Another method that is a little different from its implementation with bulk
materials, DSC can be used to determine the nature and speciation of crystallinity
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within nanoparticles through the measurement of glass and melting point temperatures
and their associated enthalpies 129.
4.5. DIARRHEAGENIC ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)
E. coli are motile Gram-negative bacteria that occupy the intestines of humans
and animals as commensals or as pathogens. The genome consists of a singular
circular DNA molecule of 4 x 106 base pairs with a molecular weight of ~ 2.5 x 109
Daltons. E. coli are sero typed based on their O lipopolysaccharide (LPS) and H
(flagellar) surface antigen (Ag) profiles. ETEC possess five different fimbrial
subtypes, F4 (K88), F18, F5 (K99), F6 (987P), and F41, F5, F6, and F41 fimbriae are
predominantly associated with ETEC that cause scours in neonatal pigs, whereas the
F4 and F18 adhesins are found on ETEC that predominantly cause post-weaning coli
bacillosis (PWC). Once colonization is established, ETEC can elaborate heat-labile
(LT), heat-stable (ST), and East-1 enterotoxins that act upon intestinal enterocytes
causing secretory diarrhea. A variant of two ETEC known as ETEC/STEC (shiga
toxin E. coli) can produce, in addition to LT and ST enterotoxins, Shiga-toxin that
allows STEC to cause edema disease 130-132.
4.5.1. Enterotoxins of ETEC F4 responsible for post-weaning diarrhea in pigs
ETEC F4 infections are responsible for significant death and morbidity in
neonatal and post-weaned pigs. ETEC expressing F4 fimbriae adhere to F4R on
intestinal epithelial cells of F4R positive pigs (F4Rpos) only, resulting in colonization
and subsequent enterotoxin secretion that leads to osmotic diarrhea in piglets.
Depending upon the strain, ETEC F4 express heat-labile (LT), heat-stable (ST), and
or EAST 1 enterotoxins which cause diarrhea 133. ETEC produce one or both of two
enterotoxins, heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). LT was
found to be similar to cholera toxin physiologically, structurally, and antigenically
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and have a similar mode of action. The molecular mass (84 kDa) and the subunit
structure of the two toxins were essentially identical, with an active (A) subunit
surrounded by five identical binding (B) subunits. Following colonization of the small
intestine by ETEC and further release of the LT, the LTB subunits bind irreversibly to
GM1 ganglioside, and the A subunit activates adenylate cyclase, which results in
increases in cyclic AMP, which stimulates chloride secretion in the crypt cells and
inhibits neutral sodium chloride in the villus tips. When these actions exceed the
absorptive capacity of the bowel, purging of watery diarrhea results 134, 135.
ST is a non antigenic low-molecular-weight peptide, consisting of 18 to 19
amino acids. There are two variants, STp and STh, named from their initial discovery
from pigs and humans, respectively, and which have identical mechanisms of action.
Released in the small intestine, ST binds reversibly to guanylate cyclase, resulting in
increased levels of cyclic GMP. ST has also been implicated in the control of cell
proliferation via elevation of intracellular calcium levels. ST is small, monomeric
toxins containing multiple cysteine residues, whose disulfide bonds were mainly
responsible for the heat stability of these toxins. As with LT, chloride secretion by the
crypt cells is then increased and inhibition of neutral sodium chloride absorption
occurs, leading to outpouring of diarrheal stool. Since ETEC must be recognized by
the enterotoxins it produces, diagnosis must depend upon identifying either LT and/or
ST 136.
4.5.2. The fimbriae of the enterobacteriaciae
Fimbriae are 0.5-1.5 microns long proteinaceous appendages peritrichously
distributed at the surface of the bacterium that allow adherence of the bacteria to
fimbriae-specific receptors. Between 100-300 fimbriae are present per bacterium.
Fimbriae are classified according to the presence and position of various amino acids
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(aa) in the primary sequence of their major fimbrial subunit. The F4 fimbriae are
placed into a class that do not contain the amino acid cysteine and contain a unique
penultimate tyrosine at their carboxyl terminal end 137. The F4 fimbriae are lectins that
bind to glycoprotein receptors located on the villi lining the small intestine of pigs.
The genes faeA to faeJ are located on a plasmid and give rise to the corresponding
proteins FaeA to FaeJ whose functions range from chaperones to building blocks for
the F4 fimbriae. The F4 has a molecular weight of approximately 26 kDa as
determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE). F4 fimbriae are ~ 1 um in length and are primarily composed of hundreds of
repeating major subunits known as FaeG with a relatively small number of minor
subunits interspersed throughout the structure. The minor subunits are not only part of
the fimbrial structure but have important functions as well. The assembly of the F4
fimbriae begins with the expression of genes faeB through faeJ, whose rate of
transcription is influenced by the level of the repressor FaeA. FaeC initiates F4
production as it acts as the nidus to which copious amounts of FaeG subunits are
added with the minor subunits FaeH and FaeF added at integral locations throughout
the structure to permit proper elongation 138.
Figure 1: Genetic organization of the F4 gene cluster; genes are represented by boxes, the designations of the genes are given in the boxes and the molecular mass of the proteins are given in kDa; a short description of the function of the different proteins is given (according to Mol et al., 1994, 139).
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4.6.Approaches to study the pathogenicity of an enteric bacterial toxin
4.6.1. In vivo assays
4.6.1.1.Ligated intestinal loops or segments.
In this approach, small intestinal (jejunal or ileal) or colonic ligated intestinal
segments are inoculated with toxin preparations or bacterial cultures and the
subsequent presence or absence of secretion is assessed at time points up to 18 h.
Nishibuchi M et al., 140 demonstrated the use of ligated segments in which isogenic
strains of Vibrio parahaemolyticus differing only in production of the thermostable
direct hemolysin (TDH) were tested in order to report the importance of TDH in
causing the secretion.
4.6.1.2.In vivo perfusion studies.
The above method was just a modification of ligated intestinal loop technique
but allows a more precise and accurate measurement of net fluid movement across the
intestinal epithelium as well as measurements of the net change in specific ions in the
intestinal fluid over time. Practically, a ligated intestinal segment is cannulated with a
multiperforated tube and cleansed by flushing prior to initiation of the experiment 141.
Samples can be analyzed sequentially for ionic content or osmolarity to measure net
changes over time using polyethylene glycol or phenolsulfonphthalein 142 as a volume
marker. The only limitation for this method is that measurements are typically made
over only a few hours.
4.6.1.3.Oral inoculation.
Another approach that came into existence is the oral inoculation that causes
diarrhea or intestinal fluid accumulation in response of to secretion of an enteric
bacterial toxin. Oral inoculation of animal models with isogenic strains can help in
establishing the importance of a toxin for the disease to occur. Isogenic strains of
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Yersinia enterocolitica have been used to establish the relevance of heat-stable
enterotoxin that contributes to the severity of disease in a rabbit animal model 143.
4.6.1.4.Ritard model.
The reversible ileal tie adult rabbit diarrheal disease model (RITARD model)
is useful in studying the pathophysiolgy of enteric bacteria in an uninterrupted
intestine. It has been widely used because iit resembles or mimics native disease than
do some of the above approaches 144. Cecum is ligated permanently and the bacteria
are injected into the intestine at a site proximal to a temporary ileal ligature. After a
short incubation period (2 h) in order to allow the infection to become established, the
ligature is removed and the animals are observed for the development of diarrhea
(days). This model has been particularly useful in studying the immune responses to
ETEC 145 and V. cholerae 146.
4.6.2. In vitro assays.
4.6.2.1.Ussing chambers.
The Ussing chamber is a valuable and in vitro experimental approach that has
been used extensively to identify the specific changes in active ion transport
stimulated by enteric bacterial toxins 147. Briefly, intestinal epithelium or polarized
monolayers of cultured intestinal epithelial cells are mounted between Lucite
chambers under conditions of ionic, osmotic, and electrical equilibrium. Ability of an
enteric toxin to stimulate anion (usually chloride) secretion and/or to inhibit NaCl
absorption, both potentially contributing to net intestinal secretion, can be easily
measured under these conditions.
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4.6.2.2.Tissue culture assays.
These assays are another experimental approach to identify the role of enteric
bacterial toxins in causing diarrhea using wide variety of non intestinal cell lines.
Changes in shape of cells or by cytotoxicity in response to treatment with the enteric
toxin will give information about the activity of the toxin. Chinese hamster ovary
(CHO) and Y-1 adrenal cells have proven useful for identifying toxins that increase
intracellular cyclic AMP levels with respect to the changes in shape. In response to an
increase in intracellular cyclic AMP levels, CHO cells elongate 148 and Y-1 adrenal
cells become round 149.
4.7.DIAGNOSIS, TREATMENT AND MANAGEMENT
4.7.1. Laboratory Assays
Since ETEC must be recognized by the enterotoxins it produces, diagnosis
must depend upon identifying either LT and/or ST. Assays such as staphylococcal
coagglutination 150, passive latex agglutination 151, immunoprecipitation in agar, and
the Biken test 152 were found to be specific but were not used widely for diagnostic
purposes. Enzyme-linked immunosorbent assays became a widely used method for
detecting LT, particularly using microtiter GM1 ganglioside methods 153, 154.
Subsequently, combined GM1 enzyme-linked immunosorbent assays for ST and LT
were developed 154, 155. During recent years DNA probes, with either radioactive or
nonradioactive detections or GM1 enzyme-linked immunosorbent assays using
monoclonal antibodies against ST or LT have been the most widely used methods for
detection of ETEC toxins 156.
The more recently developed DNA probe methods have the capacity to detect
the structural genes for toxins and CFs and thus have the advantage of detecting
ETEC from samples which have been stored for long periods of time and where
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phenotypic changes may have taken place. These procedures are more difficult to
adapt to field sites in developing countries, where laboratory facilities may be
inadequate for molecular microbiological methods. Furthermore, in some instances
ETEC CFs can only be detected by molecular but not phenotypic methods, since they
are not exposed on the bacterial surfaces due to mutation of genes required for surface
expression 157.
The treatment of diarrheal disease due to ETEC is the same as that for cholera
or any other acute secretory diarrheal disease.
4.7.2. Rehydration
Rapid rehydration using intravenous fluids (such as Ringer�s lactate) is
required initially for all patients with severe dehydration. After restoration of blood
pressure and major signs of dehydration, patients can be put on oral rehydration
solutions for the remainder of therapy.
4.7.3. Antimicrobials
The use of antimicrobials in the treatment of ETEC diarrhea is problematic,
since an etiologic diagnosis cannot be made rapidly. This differs from the treatment of
cholera, an epidemic disease, where clinical findings and rapid laboratory tests can
readily lead to correct diagnosis. It has been difficult to study the effect of
antimicrobials in children with ETEC disease and antimicrobials are not used
routinely in treatment of childhood diarrhea. Antimicrobials that have been used in
effective treatment include doxycycline, trimethoprim-sulfamethoxazole,
erythromycin, norfloxacin, ciprofloxacin, ofloxacin, azithromycin, and rifamycin.
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4.7.4. Multidrug Resistance Patterns
Due to increasing microbial resistance of ETEC, newer drugs have been used.
A fluoroquinolone such as ciprofloxacin, levofloxacin, or ofloxacin is currently the
drug of choice, since no significant resistance to these drugs has yet developed 158-159.
A newer nonabsorbed drug, rifaxamin, has also been shown to be as effective as a
fluoroquinolone and has only recently been approved for use in the United States 160.
Multidrug resistance is increasing in ETEC due to the widespread use of
chemotherapeutic agents in countries where diarrhea is endemic.
4.7.5. PREVENTION
4.7.5.1.Vaccine Development
Prevention of ETEC infection is clearly related to water and sanitation,
including food preparation and distribution. Other methods on a microscale are
presently being done: building safe-water tube wells, chlorination/filtration/heating of
drinking water, and building and improving latrines. These attempts to block
transmission are certainly effective if implemented but cannot solve the problem
quickly. Therefore, there is much interest in the development of vaccines for
prevention of ETEC disease 161.
4.7.5.2.Purified CFs and Enterotoxoids
Various purified CFs have been tested as oral immunogens but have been
considered less suitable since they are expensive to prepare and sensitive to
proteolytic degradation 162. To protect the fimbriae from degradation in the stomach,
purified CFs have been incorporated into biodegradable microspheres. An alternative
administration route that has been considered is to give an ETEC vaccine by the
transcutaneous route. Such administration of E. coli CS6 together with LT has
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induced immune responses against CS6 in about half of the volunteers and anti-LT
responses in all of them 163.
4.7.5.3.Inactivated Whole-Cell Vaccines
Another approach that has been extensively attempted is to immunize orally
with killed ETEC bacteria that express then most important CFs on the bacterial
surface together with an appropriate LT toxoid, i.e., cholera toxin B subunit or LTB
164.
4.7.5.4.Live Oral ETEC Vaccines
The potential of live ETEC vaccines has been suggested based on previous
findings in human volunteers that a live vaccine strain expressing different CSs
afforded highly significant protection against challenge with wild-type ETEC
expressing the corresponding CS factors 165.
4.8.Porous dosage forms
Researchers are now focusing on development of porous nano materials for
controlled drug delivery because of many advantages like porous structure, high
surface area, tunable pore sizes with narrow distribution and well defined surface
properties 166-167. Porous carriers is widely used in pharmaceutical as it proves to be
promising in development of novel drug delivery systems such as floating drug
delivery systems, sustained drug delivery systems and improvement of solubility of
many poorly water soluble drugs 168-171. Because of enormous porous structure these
systems allows the incorporation of variety of drugs as they get easily adsorb and
release them in a more predictable and reproducible manner. An extensive work has
been done on use of microporous, mesoprosous and nanoporous carriers as a drug
delivery tool 172-173. However liquid penetration into these systems is dependent on
both molecular and bulk property of liquid and surface property of porous medium. It
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Dept. Of Pharmaceutics, JSSCP, Mysore 38
was reported that drug release from these porous systems can be completed within
few minutes and can be varied to upto several hours or days 174-175. Drug can be
loaded in these porous materials by various methods like simple mixing, solvent
evaporation, loading under high pressure, stirring in drug solution. Porous
nanoparticles can be made up of diverse materials in a variety of different conditions
that can be targeted to different parts of body with various size ranges. These porous
nanoparticles prove to be robust tool for drug delivery and proved to be useful to
encapsulate large variety of drugs of different chemistry and molecular weight 176.
Porous nanoparticles offer various advantages like ease of flow, processing,
aerolisation and provide a paradigm shift in drug delivery. In the coming years there
is growing interest in porous carriers as a adjuvant for drug delivery systems.
Illum L et al., 65 had described variety of different types of nasal vaccine
systems to include cholera toxin, microspheres, nanoparticles, liposomes, attenuated
virus and cells and outer membrane proteins (proteosomes). He discussed work on the
use of the cationic polysaccharide, chitosan as a delivery system for nasally
administered vaccines. Several animal studies have been carried out on influenza,
pertussis and diphtheria vaccines with good results. After nasal administration of the
chitosan-antigen nasal vaccines it was generally found that the nasal formulation
induced significant serum IgG responses similar to and secretory IgA levels superior
to what was induced by a parenteral administration of the vaccine. It was found that
animals vaccinated via the nasal route with the various chitosan-antigen vaccines
were protected against the appropriate challenge. So far the nasal chitosan vaccine
delivery system has been tested for vaccination against influenza in human subjects.
Study results revealed that the nasal chitosan influenza vaccine was both effective and
protective according to the CPMP requirements.
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Dept. Of Pharmaceutics, JSSCP, Mysore 39
Eldridge JH et al., 67 have investigated the use of biodegradable and
biocompatible microspheres as a vaccine delivery system for both parenteral and
enteral immunization. Microspheres composed of poly (DL-lactide-co-glycolide)
which contained a toxoid vaccine of Staphylococcal enterotoxin B were found to
strongly potentiate the circulating anti-toxin antibody response following
intraperitoneal injection. Following oral administration, microspheres less than 10
microns in diameter were specifically taken up into the Peyer's patches of the gut-
associated lymphoid tissue, where those greater than or equal to 5 microns remained
fixed for an extended period. Microspheres less than 5 microns were disseminated
within macrophages to the mesenteric lymph nodes, blood circulation and spleen. It
was found that oral immunization with enterotoxoid-containing microspheres induced
circulating toxin-specific antibodies and a concurrent secretory IgA anti-toxin
response in saliva, gut wash fluids and bronchial-alveolar wash (BAW) fluids. In
contrast, soluble enterotoxoid was completely ineffective as an oral immunogen.
Attarki K et al., 74 have studied the encapsulation of vaccines in
biodegradable microspheres and reported them as an excellent mucosal immunogens
with a high potential for immunization against bacterial infections. They tested the
protective immunity elicited by intragastric vaccination with phosphorylcholine (PC)
encapsulated in poly (DL-lactide-co-glycolide) (DL-PLG) microspheres against
Salmonella typhimurium in a mouse model of invasive intestinal infection. Mice were
primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with
PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG
microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC
immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent
assay, was observed in the intestinal secretions after immunization with PC-loaded
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microspheres, compared to the titers of mice immunized with free PC-thyr or blank
microspheres. Control mice were primed intraperitoneally on day 1 with 15 microg of
PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same
dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA
in intestinal secretions were equivalent to those of the mice intragastrically
immunized with PC-loaded microspheres, but protection was significantly weaker,
suggesting that either the IgAs were not functional or that other immune mechanisms
are important in protection. Results proved the potential of antigen encapsulation in
DL-PLG microspheres for eliciting protective immunity against invasive intestinal
bacterial diseases and suggest that a similar strategy could be used against diseases
caused by other PC-bearing microorganisms.
Gutierro I et al., 82 have entrapped BSA in particles of different sizes (200,
500 and 1000 nm) prepared from poly (D,L-lactic-co-glycolic) acid by a double
emulsion method. The particles were given, either intranasally, orally or
subcutaneously, to Balb/c mice and the serum IgG, IgG1 and IgG2a response elicited
was compared to that obtained by the subcutaneous administration of either free
antigen, free antigen emulsified 1:1 with Freund's Complete Adjuvant (FCA), or free
antigen administered with Al(OH)(3). The administration of 1000 nm particles
generally elicited a higher serum IgG response than that obtained with the
administration of 500 or 200 nm sized nanospheres, the immune response for 500 nm
particles being similar than that obtained with 200 nm by the subcutaneous and the
oral route, and higher by the intranasal route. It was found that the route of
administration influences the serum IgG2a/IgG1 ratio after the administration of free
antigen, but not after the administration of the particles. Therefore, differences on the
total serum IgG response induced by particles of different sizes do not result in
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differences on the IgG1 or IgG2a-type immune responses, suggesting that the antigen
processing and presentation is similar in all cases tested for PLGA particles.
Sher P et al., 166 have studied the use of low density porous carriers in the
pharmaceutical applications. Response surface methodology, using 3(2) factorial
design was used to study drug adsorption on and its release patterns from microporous
polypropylene (Accurel MP 1000) in the absence of additives. Ibuprofen, as model
drug, was adsorbed on the polymer by solvent evaporation using two organic solvents
methanol (M) and dichloromethane (DCM). The amount of carrier (100 mg) and its
particle size range (250-350 microm) were kept invariant while solvent volume (X1)
and drug amount (X2) were taken as variables. It was found that drug adsorption
pattern depended on the type and amount of solvent used. DSC, XRD, FTIR and
TGA, predict crystalline nature and physical form of adsorption. SEM showed the
penetration and adsorption of the drug in and on the microporous polymer. Accurel
MP 1000 had a pore volume of 1.992 g/cm3 and surface area of 55.9855 m2/g as
detected by mercury porosimetery. On drug adsorption, pore volume ranged from
0.413 to 1.198 g/cm3 for methanol and 0.280-0.759 g/cm3 for DCM. Similarly
surface area was in the range 38.445-25.497 m2/g for methanol and 18.710-
32.528m2/g for DCM. The drug release was investigated in phosphate buffer pH 7.2.
All batches showed excellent in vitro floating property. Drug release was partial with
recovery to complete dependent on type and volume of solvent. Effect of solvent
properties shows a positive influence on drug adsorption and release.
Byrne RS et al., 173 have investigated three commercially available
microparticulate porous ceramics, N-light N3, Starlight SLK1000 and Carbolite
16/20. Starlight SLK1000 and Carbolite 16/20 were principally composed of mullite,
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while N-light N3 was principally composed of quartz. Each porous ceramic was
partly open-cell with varying porosities and pore size distributions. Using a novel
vacuum loading technique, N-light N3 was loaded with benzoic acid, sodium
benzoate and diltiazem HCl, while Starlight SLK1000 and Carbolite 16/20 were
loaded with diltiazem HCl. The drug loading was influenced by the solution
concentration and by the porosity and bulk density of the ceramic. In vitro dissolution
testing of the loaded porous microparticles showed an initial burst release of each
drug followed by sustained release. Study results revealed that drug release was
influenced by the surface pore size distribution of the ceramic and by electrostatic
interactions between the interior and exterior microparticle surfaces and the drug.
Catarina et al., 177 have extensively studied polymeric nanoparticles as
particulate carriers in the pharmaceutical and medical fields, because they show
promise as drug delivery systems as a result of their controlled- and sustained-release
properties, subcellular size, and biocompatibility with tissue and cells. Several
methods to prepare nanoparticles have been developed during the last two decades,
classified according to whether the particle formation involves a polymerization
reaction or arises from a macromolecule or preformed polymer. In this review the
most important preparation methods are described, especially those that make use of
natural polymers. This study laid emphasis on advantages and disadvantages so as to
facilitate selection of an appropriate nanoencapsulation method according to a
particular application.
Wendorf et al., 178 have prepared nanoparticles ranging in size from 110 to
230 nm using Poly (lactide-co-glycolide) (PLG) polymers. The objective of this work
was to obtain a nanoparticle formulation that could be sterile filtered, lyophilized, and
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Dept. Of Pharmaceutics, JSSCP, Mysore 43
resuspended to the initial size with excipients appropriate for use as a vaccine
formulation. Protein antigens were adsorbed to the particles; the protein-nanoparticles
were then lyophilized with the excipients. Vaccine compatible excipient combinations
of sugars alone, surfactants alone, and sugars and surfactants were tested to find
conditions where initial particle size was recovered. It was reported that nanoparticle
formulations in vivo were either similar or had enhanced immunogenicity compared
to aluminum hydroxide formulations. It is concluded that a lyophilized nanoparticle
formulation with adsorbed protein antigen and minimal excipients is an effective
vaccine delivery system.
Borges et al., 179 have studied that the adsorption of antigens onto chitosan
particles is an easy and unique mild loading process suitable to be used with vaccines.
In order to increase the stability of this particles and to prevent an immediate
desorption in gastrointestinal fluids, a coating process with sodium alginate was
developed. One of the challenges of this developing process was to keep the particles
in the nanosized range in order to be taken up by M-cells of the Peyer�s patches. The
observed inversion of the particles� zeta potential values after coating suggested the
presence of an alginate coating layer. These results were confirmed by FTIR and DSC
techniques. Additionally, in vitro release studies showed that the presence of the
alginate layer around the particles was able to prevent a burst release of loaded
ovalbumin and to improve the stability of the nanoparticles in simulated intestinal
fluid at 37 ◦C. The optimization of the coating process resulted in 35% (w/w) for the
loading capacity of the coated particles. SEM investigations confirmed a suitable size
of the coated nanoparticles for the uptake by M cells.
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Dept. Of Pharmaceutics, JSSCP, Mysore 44
Rieux et al., 180 have laid emphasis on peptides and proteins that remain
poorly bioavailable upon oral administration. One of the most promising strategies to
improve their oral delivery relies on their association with colloidal carriers, e.g.
polymeric nanoparticles, stable in gastrointestinal tract, protective for encapsulated
substances and able to modulate physicochemical characteristics, drug release and
biological behavior. The mechanisms of transport of these nanoparticles across
intestinal mucosa are reviewed. In particular, the influence of size and surface
properties on their non specific uptake or their targeted uptake by enterocytes and/or
M cells is discussed. Enhancement of their uptake by appropriate cells, i.e. M cells by
(i) modeling surface properties to optimize access to and transport by M cells (ii)
identifying surface markers specific to human M cell allowing targeting to M cells
and nanoparticles transcytosis is illustrated. Encouraging results upon in vivo testing
are reported but low bioavailability and lack of control on absorbed dose slow down
products development. It was reported that vaccines are certainly the most promising
applications for orally delivered nanoparticles.
Arayne et al., 181 have laid emphasis mainly on fabrication of porous
nanoparticles, its characterization and its use for controlled release of drug. It also
encompasses the strategies that have been used to translate and fabricate a wide range
of particulate carriers e.g., nanospheres, liposomes, micelles, oil-in-water emulsions,
with prolonged circulation and/or target specificity. Sol-gel technique is one of the
most widely used techniques to fabricate porous nanoparticles within the polymer.
Such nanoparticles have also applications in vascular drug delivery and release, site-
specific targeting, as well as transfusion medicine. This article will highlight rational
approaches in design and surface engineering of nanoscale vehicles and entities for
site-specific drug delivery.
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Dept. Of Pharmaceutics, JSSCP, Mysore 45
Huyghebaert N et al., 182 have developed a multi-particulate formulation of
F4 fimbriae for oral vaccination of suckling piglets against enterotoxigenic
Escherichia coli infections. From the economical point of view, a pellet formulation
was optimized to decrease vaccine dose and dosing frequency. It was found after
disintegration testing, pellets consisting of lactose (a-lactose monohydrate 90
mesh/lactose 75/25 (w/w)) and microcrystalline cellulose in a ratio of 80/20 (w/w)
showed a sponge-like structure from which F4 fimbriae could be released. Coating of
these pellets resulted in good enteric properties. Dissolution test showed that F4
fimbriae were released from the optimized enteric-coated pellets but interaction
between F4 fimbriae and the coating polymer was seen. This incompatibility leads to
unpredictable in vitro quantification of F4 biological activity.
Broeck W V et al., 183 have studied different procedures for preparing and
purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26
(O149:K91:F4ac LT+Sta+STb+) and the purity and yield of F4ac were compared.
Fimbriae were prepared by either mechanical shearing or heat shock treatment of
concentrated bacterial suspensions (1011 bacteria/ml). The mechanical shearing
procedure resulted in approximately 1.7 mg fimbriae (i.e. 74.4% of the isolated
protein) and 0.6 mg (25.6%) contaminating proteins per 1012 bacteria, whereas the
yield of fimbriae following heatshock treatment was lower (0.3 mg per 1012 bacteria,
i.e. 26.2%) and the relative contamination higher (1.0 mg per 1012 bacteria, i.e.
73.8%). A further purification consisted of either anion exchange chromatography
(AEC) or electro-elution from SDS-polyacrylamide gels. It was concluded that native
fimbriae as well as major subunits were able to bind to the receptors, and the
specificity of the binding was demonstrated by blockage with F4ac-specific MAb.
Review of Literature
Dept. Of Pharmaceutics, JSSCP, Mysore 46
Verdonck F et al., 184 have studied that receptor-mediated uptake of orally
administered antigen can lead to an antigen-specific immune response, whereas oral
administration of most other non-replicating soluble antigens results in the induction
of oral tolerance. In the present study, it is shown that fimbriae purified from an F4
(K88) + enterotoxigenic Escherichia coli strain can function as a mucosal carrier
molecule for the model antigen human serum albumin (HSA). Study indicates that F4
fimbriae as mucosal carrier and CT as adjuvant synergistically improve the induction
of a HSA-specific immune response following oral immunization of pigs. These
results could open new perspectives in the development of vaccines against
enteropathogens.
Broeck V W et al., 185 have developed an effective way of stimulating the
mucosal immune system which was examined in piglets, using F4 fimbriae of
enterotoxigenic Escherichia coli (ETEC). It was demonstrated that purified F4
fimbriae, as opposed to oval albumin (OVA), are powerful oral immunogens.
Furthermore, a priming of the mucosal immune system is better obtained by oral
infection (ASC localized in mesenteric LN) than by IM F4 injection (ASC localized
in spleen and retropharyngeal LN) since an oral boost with purified F4 induced a
secondary response in the orally infected animal (mainly IgA and IgG ASC, rapid
increase of IgA antibodies) while in the IM primed animal a secondary (more
circulating antigen- specific ASC than in the unprimed animal) as well as a primary
IgM and IgA response (mainly IgM ASC, slow increase of IgA antibodies),
suggesting a primary mucosal response, were seen. An oral challenge of the naive
control displayed a primary response (mainly IgM ASC, slow increase of IgA and IgG
antibodies). It was concluded that the capacity of purified F4 to activate the mucosal
Dept. Of Pharmaceutics, JSSCP, Mysore
immune system on oral administration, is of importance for the development of oral
vaccines against ETEC infections.
Verdonck F et al
available against intestinal infections since the induction of a protective intestinal
immune response is difficult to achieve. For instance, oral administration of most
proteins results in oral tolerance instead of an antigen
shown before that as a result of oral immunization of piglets with F4 fimbriae purified
from pathogenic enterotoxigenic
receptor (F4R) in the intestine and induce a protective F4
Data suggest and concluded that the mucosal immunogenicity of soluble virulence
factors can be increased by the construction of stable polymeric structures and
therefore help in the development of effective mucosal vaccines.
Murillo M et al.,
Brucella ovis by the spray
glycolide RG502H [PLGA], and blends with poly
obtain microparticles smaller than 5 ìm. Microparticles were
efficiency, release studies, acidification of the in vitro release medium, and in vitro
J744-macrophage experiments (phagocytosis and toxicity of the preparations) to
determine the optimal formulation for vaccination purposes. All
suggest that the microparticulated antigenic formulation containing the higher ratio of
PEC is susceptible to be used in animal vaccination studies.
Haining et al., 188
that exploits the ability of APCs to cross
engulfed in the low pH environment
Review o
Dept. Of Pharmaceutics, JSSCP, Mysore
immune system on oral administration, is of importance for the development of oral
vaccines against ETEC infections.
Verdonck F et al., 186 have laid emphasis on vaccines that are commercially
inal infections since the induction of a protective intestinal
immune response is difficult to achieve. For instance, oral administration of most
proteins results in oral tolerance instead of an antigen-specific immune response. It is
a result of oral immunization of piglets with F4 fimbriae purified
from pathogenic enterotoxigenic Escherichia coli (ETEC), the fimbriae bind to the F4
receptor (F4R) in the intestine and induce a protective F4-specific immune response.
ncluded that the mucosal immunogenicity of soluble virulence
factors can be increased by the construction of stable polymeric structures and
therefore help in the development of effective mucosal vaccines.
., 187 have encapsulated the antigenic extract Hot Saline from
by the spray-drying technique with different polyesters (poly
glycolide RG502H [PLGA], and blends with poly- -caprolactone [PEC]) in order to
obtain microparticles smaller than 5 ìm. Microparticles were tested for encapsulation
efficiency, release studies, acidification of the in vitro release medium, and in vitro
macrophage experiments (phagocytosis and toxicity of the preparations) to
determine the optimal formulation for vaccination purposes. All these characteristics
suggest that the microparticulated antigenic formulation containing the higher ratio of
PEC is susceptible to be used in animal vaccination studies.
8 have reported the use of a novel pH-triggered
that exploits the ability of APCs to cross-present MHC I-restricted cells that have been
engulfed in the low pH environment of the phagosome. A model MHC class I
Review of Literature
47
immune system on oral administration, is of importance for the development of oral
laid emphasis on vaccines that are commercially
inal infections since the induction of a protective intestinal
immune response is difficult to achieve. For instance, oral administration of most
specific immune response. It is
a result of oral immunization of piglets with F4 fimbriae purified
(ETEC), the fimbriae bind to the F4
specific immune response.
ncluded that the mucosal immunogenicity of soluble virulence
factors can be increased by the construction of stable polymeric structures and
enic extract Hot Saline from
drying technique with different polyesters (poly-lactide-co-
caprolactone [PEC]) in order to
tested for encapsulation
efficiency, release studies, acidification of the in vitro release medium, and in vitro
macrophage experiments (phagocytosis and toxicity of the preparations) to
these characteristics
suggest that the microparticulated antigenic formulation containing the higher ratio of
microparticle
that have been
of the phagosome. A model MHC class I-
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Dept. Of Pharmaceutics, JSSCP, Mysore 48
restricted peptide Ag from the influenza A matrix protein was encapsulated in spray-
dried microparticles composed of dipalmitoylphosphatidylcholine and the pH-
sensitive polymethacrylate Eudragit E100. Encapsulation of the peptide in the
microparticles resulted in efficient presentation of the peptide to CD8+ T cells by
human dendritic cells in vitro, and was superior to unencapsulated peptide or peptide
encapsulated in an analogous pH-insensitive particle. These microparticles can be
modified to coencapsulate a range of adjuvants along with the Ag of interest.
Encapsulation of MHC I epitopes in pH-triggered microparticles increases Ag
presentation and may improve CD8+ T cell priming to peptide vaccines against
viruses and cancer.
Baras B et al., 189 have worked on the immunogenicity of a single nasal or
oral administration of recombinant 28-kDa glutathione S-transferase of Schistosoma
mansoni (rSm28GST) entrapped by poly (lactideco- glycolide) (PLG)- or
polycaprolactone (PCL) biodegradable microparticles. Whatever the polymer and the
route of administration used, the equivalent of 100 mg of entrapped rSm28GST
induced a long-lasting and stable antigen-specific serum antibody response, with a
peak at 9 to 10 weeks following immunization. Pooled 10-week sera from mice
receiving PLG microparticles by the nasal or oral route neutralized the rSm28GST
enzymatic activity, whereas sera of mice receiving either PCL microparticles, free
rSm28GST, or empty microparticles inefficiently neutralized this enzymatic activity.
Study shows that a single administration of these microparticles could provide distinct
and timely release pulses of microencapsulated antigen, which might greatly facilitate
future vaccine development.
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Dept. Of Pharmaceutics, JSSCP, Mysore 49
Gregory et al., 190 have prepared starch based microporous microparticles by
spray drying techniques and had discussed its properties and applications.
Gupta et al., 191 have prepared prostaglandin E1 (PGE1) encapsulated large
porous microparticles for sustained pulmonary vasodilation and hypothesized that
incorporation of an effervescent porogen, ammonium bicarbonate (NH4HCO3), in
internal aqueous phase results in development of large and highly porous
microparticles ideal for deep lung deposition of the inhaled drug at a controlled rate.
Jain et al.,192 have prepared porous carrier based floating orlistat microspheres
for gastric delivery using calcium silicate as porous carrier, orlistat, an oral anti-
obesity agent; and eudragit S as polymer, by solvent evaporation method and to
evaluate their gastro-retentive and controlled-release properties. The effect of various
formulation and process variables on the particle morphology, micromeritic
properties, in vitro floating behavior, percentage drug entrapment, and in vitro drug
release was studied. The gamma scintigraphy of the optimized formulation was
performed in albino rabbits to monitor the transit of floating microspheres in the
gastrointestinal tract. The orlistat-loaded optimized formulation was orally
administered to albino rabbits, and blood samples collected were used to determine
pharmacokinetic parameters of orlistat from floating microspheres. The microspheres
were found to be regular in shape and highly porous. Microsphere formulation CS4,
containing 200 mg calcium silicate, showed the best floating ability (88% +/- 4%
buoyancy) in simulated gastric fluid as compared with other formulations. It was
found that release pattern of orlistat in simulated gastric fluid from all floating
microspheres followed Higuchi matrix model and Peppas-Korsmeyer model.
Prolonged gastric residence time of over 6 hours was achieved in all rabbits for
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Dept. Of Pharmaceutics, JSSCP, Mysore 50
calcium silicate-based floating microspheres of orlistat. The enhanced elimination
half-life observed after pharmacokinetic investigations in the present study is due to
the floating nature of the designed formulations.
Gu et al., 193 have demonstrated magnetic manipulation, fluorescent tracking,
and localized delivery of a drug payload to cancer cells in vitro using nanostructured
porous silicon microparticles as a carrier. The multifunctional microparticles are
prepared by electrochemical porosification of a silicon wafer in a hydrofluoric acid-
containing electrolyte, followed by removal and fracture of the porous layer into
particles using ultrasound. The intrinsically luminescent particles are loaded with
super paramagnetic iron oxide nanoparticles and the anti-cancer drug doxorubicin.
The drug-containing particles are delivered to human cervical cancer (HeLa) cells in
vitro, under the guidance of a magnetic field. It was found that the high concentration
of particles in the proximity of the magnetic field results in a high concentration of
drug being released in that region of the Petri dish, and localized cell death is
confirmed by cellular viability assay.
Rawat et al., 194 have investigated the feasibility of large porous particles as
long-acting carriers for pulmonary delivery of low molecular weight heparin
(LMWH). Microspheres were prepared with a biodegradable polymer, poly(lactic-co-
glycolic acid) (PLGA), by a double-emulsion�solvent-evaporation technique. The
drug entrapment efficiencies of the microspheres were increased by modifying them
with three different additives�polyethyleneimine (PEI), Span 60 and stearylamine.
The resulting microspheres were evaluated for morphology, size, zeta potential,
density, in vitro drug-release properties, cytotoxicity, and for pulmonary absorption in
vivo. Scanning electron microscopic examination suggests that the porosity of the
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Dept. Of Pharmaceutics, JSSCP, Mysore 51
particles increased with the increase in aqueous volume fraction. It was found that the
amount of aqueous volume fraction and the type of core-modifying agent added to the
aqueous interior had varying degrees of effect on the size, density and aerodynamic
diameter of the particles. Study results showed that when PEI was incorporated in the
internal aqueous phase, the entrapment efficiency was increased from 16.22 ± 1.32%
to 54.82 ± 2.79%. The amount of drug released in the initial burst phase and the
release-rate constant for the core-modified microspheres were greater than those for
the plain microspheres. After pulmonary administration, the half-life of the drug from
the PEI- and stearylamine-modified microspheres was increased by 5- to 6-fold
compared to the drug entrapped in plain microspheres. The viability of Calu-3 cells
was not adversely affected when incubated with the microspheres. It was found that
newly developed porous microspheres of LMWH have the potential to be used in a
form deliverable by dry-powder inhaler as an alternative to multiple parenteral
administrations of LMWH.
Gupta et al., 195 have investigated the hypothesis of large porous poly (lactic-
co-glycolic acid) (PLGA) microparticles modified with polyethyleneimine (PEI) are
viable carriers for pulmonary delivery of prostaglandin E1(PGE1) used in the
treatment of pulmonary arterial hypertension (PAH), a pulmonary vascular disorder.
The particles were prepared by a double-emulsion solvent evaporation method with
PEI-25 kDa in the internal aqueous phase to produce an osmotic pressure gradient.
Polyvinyl alcohol (PVA) was used for external coating of the particles. The particles
were examined for morphology, size, aerodynamic diameter, surface area, pore
volume and in-vitro release profiles. Particles with optimal properties for inhalation
were tested for in-vivo pulmonary absorption, metabolic stability in rat lung
homogenates, and acute toxicity in rat bronchoalveolar lavage fluid and respiratory
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Dept. Of Pharmaceutics, JSSCP, Mysore 52
epithelial cells, Calu-3. The micromeritic data indicated that the PEI-modified
particles of PGE1 are optimal for inhalation. It was found that incorporation of PEI in
the formulations resulted in an increased entrapment efficiency 83.26 ± 3.04% for
particles with 1% PVA and 95.48±0.46% for particles with 2% PVA. The amount of
cumulative drug released into the simulated interstitial lung fluid was between
50.8±0.76% and 55.36±0.06%. A remarkable extension of the circulation half-life up
to 6.0�6.5 hours was observed when the formulations were administered via the
lungs. The metabolic stability and toxicity studies showed that the optimized
formulations were stable at physiological conditions and relatively safe to the lungs
and respiratory epithelium. Overall, this study demonstrates that large porous
inhalable polymeric microparticles can be a feasible option for non-invasive and
controlled release of PGE1 for treatment of PAH.
Borges et al., 196 have found that the adsorption of antigens onto chitosan
particles is an easy and unique mild loading process suitable to be used with vaccines.
In order to increase the stability of this particles and to prevent an immediate
desorption in gastrointestinal fluids, a coating process with sodium alginate was
developed. One of the challenges of this developing process was to keep the particles
in the nanosized range in order to be taken up by M-cells of the Peyer�s patches. The
observed inversion of the particles� zeta potential values after coating suggested the
presence of an alginate coating layer. These results were confirmed by FTIR and DSC
techniques. In vitro release studies showed that the presence of the alginate layer
around the particles was able to prevent a burst release of loaded ovalbumin and to
improve the stability of the nanoparticles in simulated intestinal fluid at 37 ◦C. The
optimisation of the coating process resulted in 35% (w/w) for the loading capacity of
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Dept. Of Pharmaceutics, JSSCP, Mysore 53
the coated particles. SEM investigations confirmed a suitable size of the coated
nanoparticles for the uptake by M-cells.
Janet et al., 197 have prepared a nanoparticle formulation that could be sterile
filtered, lyophilized, and resuspended to the initial size with excipients appropriate for
use as a vaccine formulation. Poly (lactide-co-glycolide) (PLG) polymers were used
to create nanoparticles ranging in size from 110 to 230 nm. Protein antigens were
adsorbed to the particles; the protein-nanoparticles were then lyophilized with the
excipients. Vaccine compatible excipient combinations of sugars alone, surfactants
alone, and sugars and surfactants were tested to find conditions where initial particle
size was recovered. Sterile filtration of smaller nanoparticles led to minimal PLG
losses and allowed the particle preparation to be a nonaseptic process. We found that
the smaller nanoparticles of size -120 nm required higher surfactant concentration to
resuspend post lyophilization than slightly larger (-220 nm) particles. To resuspend
120 nm nanoparticles formulations of poly (vinyl alcohol) (PVA) with
sucrose/mannitol or dioctyl sodium sulfosuccinate (DSS) with trehalose/mannitol
were sufficient. The protein nanoparticles resuspension with the same excipients was
dependent on the protein and protein loading level. It was found that nanoparticle
formulations in vivo were either similar or had enhanced immunogenicity compared
to aluminum hydroxide formulations. A lyophilized nanoparticle formulation with
adsorbed protein antigen and minimal excipients is an effective vaccine delivery
system.
Koh et al., 198 have reported that enterotoxigenic Escherichia coli (ETEC)
infections result in large economic losses in the swine industry worldwide. The
organism causes diarrhea by adhering to and colonizing enterocytes in the small
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Dept. Of Pharmaceutics, JSSCP, Mysore 54
intestines. While much progress has been made in understanding the pathogenesis of
ETEC, no homologous intestinal epithelial cultures suitable for studying porcine
ETEC pathogenesis have been described prior to this report. In the current study, we
investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1
and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell
line was assessed for its ability to support the adherence of E. coli expressing fimbrial
adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18.Wild-type ETEC
expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2
cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine
cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E.
coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to
IPEC-J2 cells. It was found that the E. coli strains G58-1 and 711 which express no
fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine
cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The
binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities,
together with lack of binding of 987P ETEC and non-fimbriated E. coli strains,
suggests strain-specific E. coli binding to these cell lines. These findings suggest the
potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.
Moon et al., 199 have demonstrated the use of fimbrial vaccines given
parenterally to pregnant cattle, sheep and swine to protect suckling newborn calves,
lambs and pigs against enterotoxigenic Escherichia coli (ETEC) infections. Such
vaccines are practical and effective because (1) most fatal ETEC infections in farm
animals occur in the early neonatal period when the antibody titres in colostrum and
milk are highest, (2) more than 90% of the ETEC in farm animals belong to a small
family of fimbrial antigen types, (3) fimbriae consist of good protein antigens on the
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Dept. Of Pharmaceutics, JSSCP, Mysore 55
bacterial surface where the)' are readily accessible to antibody, (4)fimbriae are
required or a critical step (adhesion-colonization) early in the pathogenesis of the
disease. ETEC infections continue to be a significant clinical problem in farm
animals' in spite of extensive use of fimbriae-based vaccines. Definitive data on the
efficacy of the commercial vaccines infield use are not available. The prevailing
perception among animal health professionals is that the vaccines are effective, that
the problem occurs chiefly among non-vaccinated animals and that in some herds
vaccination moves peak prevalence of disease from the first to the second or third
week after birth, when mortality is lower. It has been suggested that extensive use of
vaccines will rapidly select for the emergence of novel or previously low prevalence
fimbrial antigen types. There is no evidence that this has happened after a decade of
routine vaccine use in the United States. However, there is no active direct
surveillance for such emergence. In contrast to the rational development of vaccines
to provide passive lacteal protection against ETEC in suckling neonates,
comparatively little progress has been made in providing the knowledge required .for
development of vaccines to protect against postweaning ETEC infections in swine.
Li et al., 200 had reported that escherichia coli expressing F4 fimbriaeis the
major pathogenic bacteria that causes diarrhea in piglets before weaning. The
adhesion of E. coli to the brush borders of the epithelial cells of piglets is the
precondition leading to diarrhea, which in turn is due to the presence of the F4
receptors determined by an autosomal recessive gene on the brush borders of the
epithelial cells. In order to clarify the genetic mechanism of the adhesion, an in vitro
adhesion experiment was carried out for three variants of E. coli F4 (ab, ac, and ad) in
366 piglets of three pig breeds [Landrace (LR), Large White (LW), and Songliao
Black (SB)]. The results showed that there existed significant differences (P<0.001)
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Dept. Of Pharmaceutics, JSSCP, Mysore 56
in the adhesion percentage among the three breeds. Most SB piglets were nonadhesive
for all the three variants, whereas most LR piglets were adhesive. Within each breed
except for LR, the propor-tions of the three F4 variants adhering to the brush borders
differed significantly. According to the patterns of the adhesion of the three F4
variants in the three breeds, it is very likely that the three F4 variants F4ab, F4ac, and
F4ad have different receptors that are controlled by three different loci.
Verdonck et al., 201 have reported that only a few vaccines are commercially
available against intestinal infections since the induction of a protective intestinal
immune response is difficult to achieve. For instance, oral administration of most
proteins results in oral tolerance instead of an antigen-specific immune response. It
was shown before that as a result of oral immunization of piglets with F4 fimbriae
purified from pathogenic Enterotoxigenic Escherichia coli (ETEC), the fimbriae bind
to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune
response. F4 fimbriae are very stable polymeric structures composed of some minor
subunits and amajor subunit FaeG that is also the fimbrial adhesin. In the present
study, the mutagenesis experiments identified FaeG amino acids 97 (N to K) and 201
(I to V) as determinants for F4 polymeric stability. The interaction between the FaeG
subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae
behaved identically in F4R binding and showed equal stability in the gastro-intestinal
lumen. Oral immunization experiments indicated that a higher degree of
polymerisation of the fimbriae in the intestinewas correlated with a better F4-specific
mucosal immunogenicity. These data suggest that the mucosal immunogenicity of
soluble virulence factors can be increased by the construction of stable polymeric
structures and therefore help in the development of effective mucosal vaccines.
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Dept. Of Pharmaceutics, JSSCP, Mysore 57
Snoeck et al., 202 have developed oral immunization model in pigs in which
F4 (K88) fimbriae of enterotoxigenic Escherichia coli are administered to induce a
protective intestinal immunity, was used to determine the optimal inductive sites of
the F4-specific intestinal immune response. Hereto, pigs were immunised with F4
orally, in the lumen of the mid-jejunum, ileum or mid-colon. Throughout the small
intestine, the highest number of ASC was found following jejunal immunisation,
followed by ileal, oral and colonic immunisation. To determine the significance of
Peyer�s patches in the induced immune response, F4 was injected into the jejunal
Peyer�s patches (JPP), lamina propria (LP) and ileal Peyer�s patches (IPP).
Immunisation in the JPP induced the highest number ASC in the small intestine,
whereas immunisation in the LP and IPP resulted in lower intestinal antibody
responses. In conclusion, we have shown that the JPP are the major inductive sites of
the F4-specific intestinal antibody response. This knowledge could be important when
using the pig as an animal model for vaccination studies.
Fairbrother et al., 203 have reported that escherichia coli is one of the most
important causes of postweaning diarrhea in pigs. This diarrhea is responsible for
economic losses due to mortality, morbidity, decreased growth rate, and cost of
medication. The E. coli causing postweaning diarrhea mostly carry the F4 (K88) or
the F18 adhesin. Recently, an increase in incidence of outbreaks of severe E. coli-
associated diarrhea has been observed worldwide. The factors contributing to the
increased number of outbreaks of this more severe form of E. coli-associated diarrhea
are not yet fully understood. These could include the emergence of more virulent E.
coli clones, such as the O149:LT:STa:STb:EAST1:F4ac, or recent changes in the
management of pigs. Development of multiple bacterial resistance to a wide range of
commonly used antibiotics and a recent increase in the prevalence and severity of the
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Dept. Of Pharmaceutics, JSSCP, Mysore 58
postweaning syndromes will necessitate the use of alternative measures for their
control. New vaccination strategies include the oral immunization of piglets with live
avirulent E. coli strains carrying the fimbrial adhesins or oral administration of
purified F4 (K88) fimbriae. Other approaches to control this disease include
supplementation of the feed with egg yolk antibodies from chickens immunized with
F4 or F18 adhesins, breeding of F18- and F4-resistant animals, supplementation with
zinc and/or spray-dried plasma, dietary acidification, phage therapy, or the use of
probiotics. To date, not a single strategy has proved to be totally effective and it is
probable that the most successful approach on a particular farm will involve a
combination of diet modification and other preventive measures.
Deprez et al., 204 have studied the post weaning excretion rate of hemolytic E.
coli was determined in piglets from herds affected with edema disease and on control
farms. No distinct difference in rate of excretion was observed. A split litter trial was
set up to evaluate the importance of sow's milk in the postweaning rise of fecal
hemolytic E. coli. 525 ml of sow's milk a day, mixed with the feed, completely
inhibited that postweaning rise, even after oral challenge with a pathogenic strain.
Rasschaert et al., 205 have analyzed quantitatively the mucin 4 polymorphism
for determining the F4ac/ab receptor status of a total of 63 pigs by comparing it with
the in vitro villous adhesion assay. The probability of a susceptible genotype for the
mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250
microm villi (P=0.029 for F4ab, P=0.030 for F4ac), with the odds ratio for each unit
increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004-1.069]) and
1.018 (95% CI [1.002-1.034]). In the phenotypic in vitro villous adhesion test, a cut-
off value of 5 bacteria was chosen as criteria for the distinction between an F4R
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Dept. Of Pharmaceutics, JSSCP, Mysore 59
positive and F4R negative pig. The sensitivity and specificity for the in vitro villous
adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and
24%, respectively, for F4ab as well as F4ac. Absence of adhesion of F4ac and F4ab
ETEC to the villous brush borders was not associated with genotypic resistance
suggesting that there is at least one other receptor for F4ab/ac Escherichia coli. As a
consequence, not only mucin 4 gene polymorphism but also expression of these other
receptor(s) has to be included in a screening assay for F4ac/ab receptor negative pig.
Verdonck et al., 206 have reported that piglets are susceptible to F4 (K88) +
enterotoxigenic Escherichia coli (ETEC)-induced neonatal and post-weaning
diarrhea. The F4 fimbriae are composed of some minor subunits and the major
subunit FaeG that also constitutes the adhesin. Parenteral vaccination of sows with an
F4-containing vaccine protects the suckling piglets against neonatal F4+ ETEC-
induced diarrhea, but no commercial (mucosal) vaccine exists against F4+ ETEC-
induced weaning diarrhea. To develop a vaccine, a bioactive F4-receptor (F4R)
binding FaeG molecule is required that binds to the F4R following oral immunization
and induces a FaeG-specific immune response. The present study reports the altered
binding of the FaeG-specific monoclonal antibody IMM01 with bioactive versus non-
bioactive F4 fimbrial adhesin FaeG. The correlation of altered IMM01 binding with
altered FaeG bioactivity permits the use of an IMM01-based ELISA as a fast, specific
and sensitive in vitro selection for potent F4 or (recombinant) FaeG antigen
formulations, useful in an F4+ ETEC vaccine.
Broeck et al., 207 have demonstrated an effective way of stimulating the
mucosal immune system was examined in piglets, using F4 fimbriae of
enterotoxigenic Escherichia coli (ETEC). It was demonstrated that purified F4
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Dept. Of Pharmaceutics, JSSCP, Mysore 60
fimbriae, as opposed to ovalbumin (OVA), are powerful oral immunogens. Indeed,
oral administration of purified F4 induced antigen-specific antibody-secreting cells
(ASC) in the Peyer's patches, mesenteric lymph nodes (LN), blood and lamina propria
4, 7, 9 and 11 days postimmunization, respectively, indicating a stimulation of the
mucosal immune system, whereas upon oral administration of OVA, no immune
response was observed. Moreover, the induced F4-specific IgA and IgG antibody
responses were comparable with those obtained upon oral infection with viable E. coli
and intramuscular (i.m.) F4 injection, respectively. Furthermore, a priming of the
mucosal immune system is better obtained by oral infection (ASC localized in
mesenteric LN) than by i.m. F4 injection (ASC localized in spleen and
retropharyngeal LN) since an oral boost with purified F4 induced a secondary
response in the orally infected animal (mainly IgA and IgG ASC, rapid increase of
IgA antibodies) while in the i.m. primed animal a secondary (more circulating
antigen-specific ASC than in the unprimed animal) as well as a primary IgM and IgA
response (mainly IgM ASC, slow increase of IgA antibodies), suggesting a primary
mucosal response, were seen. An oral challenge of the naive control displayed a
primary response (mainly IgM ASC, slow increase of IgA and IgG antibodies). It was
found that the capacity of purified F4 to activate the mucosal immune system on oral
administration is of importance for the development of oral vaccines against ETEC
infections.
Broeck et al., 208 have demonstrated F4 receptor-positive (F4R+) and F4
receptor-negative (F4R-) pigs by orally vaccinating them with purified F4 fimbriae of
enterotoxigenic Escherichia coli (ETEC). Serum immunoglobulin G (IgG) and IgA
responses were readily detected in F4R+ animals, whereas immune responses were
not detected in F4R- animals. Even after a subsequent oral infection with virulent
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Dept. Of Pharmaceutics, JSSCP, Mysore 61
F4(+) ETEC and a booster immunization with F4, the F4R- animals remained F4
seronegative whereas the unvaccinated F4R+ pigs exhibited clear IgA and IgG
responses. These results clearly demonstrate that F4Rs are a prerequisite for an
immune response following oral immunization. Furthermore, indications that oral F4
vaccination can induce mucosal protection were obtained, since the experimental
ETEC infection did not induce a systemic booster response or fecal ETEC excretion
in orally vaccinated F4R+ pigs, in contrast to the clear immune response and ETEC
excretion of unvaccinated F4R+ animals. F4-specific IgA antibodies could be found
in the feces of the vaccinated F4R+ pigs. They are secreted at the intestinal mucosal
surface and appear to prevent ETEC infection. It was reported that F4R-dependent
induction of a mucosal immune response can be used as a model to better understand
mucosal immunization and mucosal immune responses and can contribute to the
development of oral vaccines in veterinary as well as in human medicine.
Rasschaert et al., 209 have quantitatively analyzed the mucin 4 polymorphism
for determining the F4ac/ab receptor status of a total of 63 pigs by comparing it with
the in vitro villous adhesion assay. The probability of a susceptible genotype for the
mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250
mm villi (P = 0.029 for F4ab, P = 0.030 for F4ac), with the odds ratio for each unit
increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004�1.069]) and
1.018 (95% CI [1.002� 1.034]). In the phenotypic in vitro villous adhesion test, a cut-
off value of 5 bacteria was chosen as criteria for the distinction between an F4R
positive and F4R negative pig. The sensitivity and specificity for the in vitro villous
adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and
24%, respectively, for F4ab as well as F4ac. It was reported that not only mucin 4
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Dept. Of Pharmaceutics, JSSCP, Mysore 62
gene polymorphism but also expression of these other receptor(s) has (have) to be
included in the screening assay for F4ac/ab receptor negative pigs.
Devriendt et al.,210 have demonstrated in his study that polymeric fimbriae
are essential for adhesion to porcine intestinal epithelial cells (IEC) and the secretion
of IL-6 and IL-8 by IEC. Infections with F4 (+) enterotoxigenic Escherichia coli
(ETEC) cause severe diarrhea in piglets, resulting in morbidity and mortality. F4
fimbriae are the key virulence factors mediating the attachment of F4 (+) ETEC to the
intestinal epithelium. Intestinal epithelial cells (IEC) are recently being recognized as
important regulators of the intestinal immune system through the secretion of
cytokines. Results of the study revealed that a potential mucosal adjuvant capacity of
ETEC-derived flagellin and may improve rational vaccine design against F4 (+)
ETEC infections.
Verdonck et al.,211 emphasized on developing vaccine for the treatment of
enterotoxigenic E. coli (ETEC) infection. Fimbriae-specific antibody response of
newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-
IIv) producing F18+ verotoxigenic E. coli (VTEC) (strain 107/86) was compared with
the response following an infection with LT producing F4+ enterotoxigenic E. coli
(ETEC) (strain GIS 26). It was reported that F4+ ETEC were able to colonize the gut
very soon after infection, as peak excretion of F4+ E. coli bacteria was seen 2 days
post-infection (dpi), but had already disappeared 7 dpi. On the other hand, F18+
VTEC infection resulted in a slower colonization of the gut as the peak excretion of
F18+ E. coli was observed between 3 and 5 dpi, but this colonization remained longer
as F18+ E. coli were detected till 9 dpi in feces. Furthermore, this fast colonization
pattern of F4+ ETEC is accompanied with the presence of F4-specific antibodies in
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Dept. Of Pharmaceutics, JSSCP, Mysore 63
mucosal tissues and serum from 4 dpi onward, with maximal amounts of F4-specific
IgA in the jejunal lamina propria and serum 7 dpi.
Verdonck et al.,212 have reported the importance of adhesins in the
pathogenicity of several bacteria on their usefulness in vaccines. Gene of the F4
(K88)-fimbrial adhesin FaeG of the pathogenic enterotoxigenic Escherichia coli
(ETEC) strain GIS26 was cloned in the pET30Ek-LIC vector and expressed with an
N-terminal His- and S-tag in the cytoplasm of BL21 (DE3). Recombinant FaeG
(rFaeG) subunits were isolated from insoluble cytoplasmic aggregates and refolded
into a native-like F4 receptor (F4R)-binding conformation. Indeed, the presence of
conformational epitopes was shown by ELISA and the ability to bind the F4R was
observed by inhibiting the adhesion of F4+ ETEC to F4R+ villi with increasing
concentrations of native-like re-folded rFaeG subunits. The rFaeG subunits appear as
monomers, whereas the purified F4 fimbriae are multimers. It was reported that oral
immunization of newly weaned piglets with native-like rFaeG induced a mucosal and
systemic F4-specific immune response, significantly reducing F4+ E. coli excretion
from 2 till 5 days following challenge infection. However, improvement of stability
and immunogenicity of rFaeG is necessary since a higher F4-specific response was
obtained following immunization with purified F4 fimbriae. Furthermore, the N-
terminal fusion of a His- and S-tag was not detrimental for binding the F4R,
supporting the use of FaeG as mucosal carrier. Results of the study revealed that oral
immunization with a recombinant fimbrial adhesin subunit of Escherichia coli
induces a mucosal and systemic fimbriae-specific immune response.
Rasschaert et al.,213 have demonstrated the internalization of F4 fimbriae in
the porcine intestinal epithelial cell line IPEC-J2. Enterotoxigenic Escherichia coli
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Dept. Of Pharmaceutics, JSSCP, Mysore 64
(ETEC) cause severe diarrhea in neonatal and recently weaned piglets. It was reported
that oral immunization of F4 receptor positive piglets with purified F4 fimbriae
induces a protective F4-specific intestinal immune response. However, in F4 receptor
negative animals no F4-specific immune response can be elicited, indicating that the
induction of an F4-specific mucosal immune response upon oral immunization is
receptor-dependent. Although F4 fimbriae undergo transcytosis across the intestinal
epithelium in vivo, the endocytosis pathways used remain unknown. The results in
the present study demonstrate that F4 fimbriae are internalized through a clathrin-
dependent pathway. It was reported that F4 fimbriae are transcytosed across
differentiated IPEC-J2 cells. This receptor-dependent transcytosis of F4 fimbriae may
explain the immunogenicity of these fimbriae upon oral administration in vivo.
Dept. Of Pharmaceutics, JSSCP, Mysore
4.9.5-Fluorouracil 214, 215
Figure 2: Structure of 5
Molecular formula
Molecular weight
Melting point
Category
Description
Dissociation Constant (pK
Solubility
Storage
Formulations
Identification
pH
Mechanism of action
Pharmacokinetic profile of 5
Half life
Volume of distribution
Clearance
Protein binding
Therapeutic uses
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Dept. Of Pharmaceutics, JSSCP, Mysore
5
Figure 2: Structure of 5-fluorouracil.
: C4H3FN2O2
: 130.1
: 282° to 283° C
: Anti-cancer.
: White to practically white, odourless, crystalline
powder
Dissociation Constant (pKa ): : 8.0
: Sparingly soluble in water, slightly soluble in
alcohol, practically insoluble in CHCl
: Stored in air tight containers
light.
: Intravenous route, capsules.
: Light absorption in the range 200
µg/ml solution in 7.4 pH buffer
absorption maximum at 267 nm
: 4.5 to 5.0
: The nucleotide of 5-FU, 5-Fluoro
thymidylate synthase blocks the conversion of
deoxyuridic acid to deoxy thymidylic acid
Pharmacokinetic profile of 5-FU
: 15 to 20 min
: 17.5 L/70kg
: 63 L/h
: 94%
: Anti neoplastic and immunosuppressive agent
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65
practically white, odourless, crystalline
Sparingly soluble in water, slightly soluble in
alcohol, practically insoluble in CHCl3
Stored in air tight containers protected from
Light absorption in the range 200-400 nm of a 10
µg/ml solution in 7.4 pH buffer exhibits
Fluoro-2-deoxy-
thymidylate synthase blocks the conversion of
deoxyuridic acid to deoxy thymidylic acid
Anti neoplastic and immunosuppressive agent
Dept. Of Pharmaceutics, JSSCP, Mysore
4.10. Metoprolol tartrate
Figure3
Molecular formula
Molecular weight
Melting point
IUPAC Name
Category
Dose
Description
Solubility
Formulations
Pharmacokinetic profile
Half life
Absorption
Bioavailability
Therapeutic uses
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Dept. Of Pharmaceutics, JSSCP, Mysore
Metoprolol tartrate 216
Figure3: Structure of Metoprolol tartarate.
: (C15H25NO3)2,C4H6O6
: 684.82.
: 282° to 283° C
: Anti-cancer.
: Beta- adrenoceptor antagonist.
: 100 to 450 mg daily, in divided dos
dose should not exceed 100 mg daily
: White crystalline powder.
: Very soluble in water; freely soluble in ethanol
(95%), in Chloroform and in dichloromethane;
slightly soluble in acetone; practically insoluble
in ether.
: Metoprolol tablets, Metoprolol SR tablets,
Metoprolol ER Capsules.
Pharmacokinetic profile
Half life : 3-4 hrs
Absorption : Completely absorbed from the GI tract.
Bioavailability : Oral bioavailability is about 40 - 50%.
Therapeutic uses : It is an effective â- adrenoceptor antagonist, used
in the treatment of angina, hypertension and
myocardial infarction.
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66
100 to 450 mg daily, in divided doses; the initial
exceed 100 mg daily.
Very soluble in water; freely soluble in ethanol
(95%), in Chloroform and in dichloromethane;
slightly soluble in acetone; practically insoluble
Metoprolol SR tablets,
the GI tract.
50%.
adrenoceptor antagonist, used
in the treatment of angina, hypertension and
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Dept. Of Pharmaceutics, JSSCP, Mysore 67
4.11. Eudragit S-100 217
Eudragit S100 is methacrylic acid copolymers are anionic co-polymeric
products of methacrylic acid and methyl methacrylates and other neutral methacrylic
acid esters. The ratio of the free carboxyl groups to the ester groups is approx. 1:2 in
EUDRAGIT® S 100.
CH3 CH3 CH3 CH3 CH3
| | | | | - [----C------------CH2------------C------------CH2-------------C----] N-
| | | | | COOH COOCH3 COOH COOCH3 COOH
Figure 4: Structure of Eudragit S-100.
Non-proprietary name : Methacrylic acid copolymer.
Synonyms : Eudragit, polymeric methacrylates
Description : white free flowing powders with at least 95% dry
polymer.
Molecular weight : ≥ 100000
Chemical name : Poly methacrylic acid, methyl methacrylates
Viscosity : 50-200 mPas
Solubility : Soluble in acetone, alcohol and 1N NaOH and
insoluble in dichloromethane, water, ethyl acetate
and petroleum ether.
Density : 0.83-0.85 g/cm3
Applications : Eudragit S100 is used as enteric coating agents
since they are resistance to gastric fluid also used
as a binder in both aqueous and organic weight
granulation processes. Also used to form the
matrix layers of transdermal delivery system and
It is soluble in > pH 7.0.
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Dept. Of Pharmaceutics, JSSCP, Mysore 68
4.12. Eudragit L 100 218
Eudragit® L 100 is anionic copolymers based on methacrylic acid and methyl
methacrylate ratios. The ratio of free carboxyl groups to ester groups is approximately
1:1 in Eudragit® L 100.
Chemical structure
Figure 5: Structure of Eudragit L-100.
Average molecular weight : Approx. 135,000 g/mol
Chemical/IUPAC name : Poly (methacrylic acid-co-methyl methacrylate)
1:1
Targeted drug release area : Jejunum
Dissolution Dissolution above pH 6.0
Description : White free-flowing powder with a faint
characteristic odour.
Functional Category : Enteric coating polymer and for preparation of
controlled release dosage form
Solubility : Practically insoluble in water, freely soluble in
ethanol and in 2-propanol, practically insoluble
in ethyl acetate. It is freely soluble in a 40 g/l
solution of sodium hydroxide.
Particle size: : At least 95 % less than 0.25 mm.
Film formation : Place 1ml of the solution prepared for the
viscosity test on a glass plate and allow drying. A
clear brittle film is formed.
Storage : Protect from warm temperature. Protect against
moisture.
Dept. Of Pharmaceutics, JSSCP, Mysore
4.13.Chitosan 219
Synonyms
2-Amino-2-deoxy
glucosamine.
Chemical name
Poly-ß-(1,4)-2-Amino-2-deoxy
Chitosan is natural polysaccharide compr
and N- acetyl glucosamine and can be derived by partial deacetylation of chitin
from crustacean shells. Chitin is second most abund
cellulose.
Structural formulae
Functional category
Coating agent; disintegrant; film forming agent; mucoadhesive; tablet binder;
viscosity increasing agent.
Description
Chitosan occurs as odorless, white or cream white powder or flakes.
Properties
Chitosan is a cationic polyamine with a high charge density at pH<6.5; and so
adheres to negatively charged surfaces and chelates metal ions. It is linear
polyelectrolyte with reactive with reactive hydroxyl and amino group. The presence
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Dept. Of Pharmaceutics, JSSCP, Mysore
deoxy-(1,4)-ß-D-glucopyranan; deacetylated chitin; poly
deoxy-d-glucose
Chitosan is natural polysaccharide comprising of copolymers of glucosamine
acetyl glucosamine and can be derived by partial deacetylation of chitin
from crustacean shells. Chitin is second most abundant natural polysacchride after
Figure 6: Structure of Chitosan.
Coating agent; disintegrant; film forming agent; mucoadhesive; tablet binder;
viscosity increasing agent.
Chitosan occurs as odorless, white or cream white powder or flakes.
Chitosan is a cationic polyamine with a high charge density at pH<6.5; and so
adheres to negatively charged surfaces and chelates metal ions. It is linear
polyelectrolyte with reactive with reactive hydroxyl and amino group. The presence
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69
glucopyranan; deacetylated chitin; poly-d-
ising of copolymers of glucosamine
acetyl glucosamine and can be derived by partial deacetylation of chitin
ant natural polysacchride after
Coating agent; disintegrant; film forming agent; mucoadhesive; tablet binder;
Chitosan occurs as odorless, white or cream white powder or flakes.
Chitosan is a cationic polyamine with a high charge density at pH<6.5; and so
adheres to negatively charged surfaces and chelates metal ions. It is linear
polyelectrolyte with reactive with reactive hydroxyl and amino group. The presence
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Dept. Of Pharmaceutics, JSSCP, Mysore 70
of a number of amino group, allows chitosan to react chemically with anionic system.
Almost all functional properties of chitosan depend on the chain length, charge
density, and charge distribution.
Acidity/alkalinity
pH = 4.0-6.0 (1 % w/v aqueous solution)
Moisture content
Chitosan absorbs moisture from atmosphere depending on initial moisture
content and temperature and humidity of surrounding air.
Properties of Chitosan:
Physical Properties:
Particle size <30 nm
Density 1.35 � 1.49 g/cc
Solubility: Insoluble in water but soluble in acids.
Chemical properties:
Cationic polyamine
High charge density at pH < 6.5
Adheres to negatively charged surfaces.
Forms gel with polyanions.
High molecular weight, linear polyelectrolyte
Incompatibilities:
Chitosan is incompatible with strong oxidizing agents.
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Dept. Of Pharmaceutics, JSSCP, Mysore 71
4.14.Triethyl citrate 219 Nonproprietary Names
BP: Triethyl citrate; PhEur: Triethylis citras; USPNF: Triethyl citrate
Synonyms
Citric acid, ethyl ester; Citroflex 2; Citrofol AI; E1505; Hydagen CAT; TEC.
Chemical Name
2-Hydroxy-1,2,3-propanetricarboxylic acid, triethyl ester
Figure 7: Structure of Triethyl citrate.
Empirical Formula and Molecular Weight
C12H20O7 276.29
Functional Category
Plasticizer.
Description
Triethyl citrate is a clear, odorless, practically colorless, oily liquid.
Typical Properties
Acid value: 0.02
Boiling point: 288°C (decomposes)
Flash point: 155°C
Pour point: -45°C
Solubility: soluble 1 in 125 of peanut oil, 1 in 15 of water.
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Dept. Of Pharmaceutics, JSSCP, Mysore 72
Miscible with ethanol (95%), acetone, and propan-2-ol.
Viscosity (dynamic): 35.2 mPa s (35.2 cP) at 25°C.
Stability and Storage Conditions
Triethyl citrate should be stored in a closed container in a cool, dry location.
When stored in accordance with these conditions, triethyl citrate is a stable product.
Applications in Pharmaceutical Formulation or Technology
Triethyl citrate and the related esters acetyltriethyl citrate, tributyl citrate, and
acetyltributyl are used to plasticize polymers in formulated pharmaceutical coatings.
The coating applications include capsules, tablets, beads, and granules for taste
masking, immediate release, sustainedrelease, and enteric formulations. Triethyl
citrate is also used as a direct food additive for flavoring, for solvency, and as a
surface active agent.
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Dept. Of Pharmaceutics, JSSCP, Mysore 73
4.15.Polyvinyl Alcohol 220, 221 Nonproprietary Names:
PhEur: Poly (Vinyl Alcohol): USP: Polyvinyl Alcohol
Synonyms
Airvol; Alcotex; Celvol; Elvanol; Gelvatol; Gohsenol; Lemol; Mowiol; poly(alcohol
vinylicus); Polyvinol; PVA; vinyl alcohol polymer.
Chemical Name
Ethenol, homopolymer
Empirical Formula and Molecular Weight
(C2H4O)n 20 000�200 000
Polyvinyl alcohol is a water-soluble synthetic polymer represented by the formula
(C2H4O)n. The value of n for commercially available materials lies between 500 and
5000, equivalent to a molecular weight range of approximately 20 000�200 000.
Figure 8: Structure of Polyvinyl Alcohol.
Description
Polyvinyl alcohol occurs as an odorless, white to cream-colored granular powder.
Typical Properties
Melting point
228°C for fully hydrolyzed grades;
180�190°C for partially hydrolyzed grades.
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Dept. Of Pharmaceutics, JSSCP, Mysore 74
Solubility
Soluble in water; slightly soluble in ethanol (95%); insoluble in organic
solvents. Dissolution requires dispersion (wetting) of the solid in water at room
temperature followed by heating the mixture to about 908C for approximately 5
minutes. Mixing should be continued while the heated solution is cooled to room
temperature
Applications in Pharmaceutical Formulation or Technology
Polyvinyl alcohol is used primarily in topical pharmaceutical and ophthalmic
formulations It is used as a stabilizing agent for emulsions (0.25�3.0% w/v).
Polyvinyl alcohol is also used as a viscosity-increasing agent for viscous
formulations such as ophthalmic products. It is used in artificial tears and contact lens
solutions for lubrication purposes, in sustained-release formulations for oral
administration and in transdermal patches. Polyvinyl alcohol may be made into
microspheres when mixed with a glutaraldehyde solution.
Stability and Storage Conditions
Polyvinyl alcohol is stable when stored in a tightly sealed container in a cool,
dry place. Aqueous solutions are stable in corrosion resistant sealed containers.
Preservatives may be added to the solution if extended storage is required. Polyvinyl
alcohol undergoes slow degradation at 100°C and rapid degradation at 200°C; it is
stable on exposure to light.
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Dept. Of Pharmaceutics, JSSCP, Mysore 75
4.16.Lutrol F68 222-223 Nonproprietary Names
BP: Poloxamers; PhEur: Poloxamera; USPNF: Poloxamer 188
Synonyms
Lutrol; Monolan; Pluronic; poloxalkol; polyethylene�propylene glycol copolymer;
polyoxyethylene�polyoxypropylene copolymer; Supronic; Synperonic.
Chemical Name
á-Hydro-ù-hydroxypoly(oxyethylene)poly(oxypropylene) poly(oxyethylene) block
copolymer
The poloxamer polyols are a series of closely related block copolymers of
ethylene oxide and propylene oxide conforming to the general formula
HO(C2H4O)a(C3H6O)b(C2H4O)aH.
Figure 9: Structure of LUTROL F68.
Description
Poloxamers generally occur as white, waxy, free-flowing prilled granules, or as cast
solids. They are practically odorless and tasteless. At room temperature, poloxamer
124 occurs as a colorless liquid.
Functional Category
Dispersing agent; emulsifying and coemulsifying agent; solubilizing agent; tablet
lubricant; wetting agent.
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Dept. Of Pharmaceutics, JSSCP, Mysore 76
Typical Properties
Acidity/alkalinity: pH = 5.0�7.4 for a 2.5% w/v aqueous solution
Density: 1.06 g/cm3 at 25°C
Flash point: 260°C
Flowability: solid poloxamers are free flowing.
HLB value: 0.5�30; 29 for poloxamer 188.
Melting point: 52�57°C
Applications in Pharmaceutical Formulation or Technology
Poloxamers are nonionic polyoxyethylene�polyoxypropylene copolymers used
primarily in pharmaceutical formulations as emulsifying or solubilizing agents. The
polyoxyethylene segment is hydrophilic while the polyoxypropylene segment is
hydrophobic. Poloxamers are used as emulsifying agents in intravenous fat
emulsions, and as solubilizing and stabilizing agents to maintain the clarity of elixirs
and syrups. Poloxamers may also be used as wetting agents; in ointments, suppository
bases, and gels; and as tablet binders and coatings. Poloxamer 188 has also been used
as an emulsifying agent for fluorocarbons used as artificial blood substitutes and in
the preparation of solid-dispersion systems. Therapeutically, poloxamer 188 is
administered orally as a wetting agent and stool lubricant in the treatment of
constipation; it is usually used in combination with a laxative such as danthron.
Poloxamers may also be used therapeutically as wetting agents in eye-drop
formulations, in the treatment of kidney stones, and as skin-wound cleansers.
Stability and Storage Conditions
Poloxamers are stable materials. Aqueous solutions are stable in the presence of acids,
alkalis, and metal ions. However, aqueous solutions support mold growth. The bulk
material should be stored in a well-closed container in a cool, dry place.
Dept. Of Pharmaceutics, JSSCP, Mysore
4.17.Ammonium carbonate
Chemical formula
CH6N2
CH8N2
CH5NO
Structural
formula NH2COONH(NH4)2
NH4HCO
Figure 10
Description White powder or hard, white or translucent masses of crystals with an odor
ammonia. On exposure to air
porous lumps or powder.
Functional uses: acidity regulator, raising agent Typical Properties
Solubility: soluble in water
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Dept. Of Pharmaceutics, JSSCP, Mysore
Ammonium carbonate 224
2O2,
2O3 NO3
COONH4
2HCO3 HCO3
Figure 10: Structure of Ammonium carbonate.
White powder or hard, white or translucent masses of crystals with an odor
ammonia. On exposure to air it become opaque and is finally converted into white
porous lumps or powder.
acidity regulator, raising agent
Solubility: soluble in water
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77
White powder or hard, white or translucent masses of crystals with an odor of
it become opaque and is finally converted into white
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Dept. Of Pharmaceutics, JSSCP, Mysore 78
4.18. Strain
ATCC® Number : 35401� 225
Organism : Escherichia coli (Migula) Castellani and Chalmers
Designations : H10407
Isolation : human feces
Depositor : S Formal
History : ATCC <<--S Formal<<--H.R. DuPont
Biosafety Level : 2
Shipped : freeze-dried
Growth Conditions : ATCC medium3: Nutrient agar or nutrient broth
Temperature: 37°C
Atmosphere: Aerobic
Antigenic Properties: serotype O78:H11
Applications: detection of LT toxin
detection of ctx A11
produces heat stable toxin
production of heat labile and heat stable
enterotoxins