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D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 1
Chapter 1:
General Introduction and Objectives of the Study
1.1. Pellets as solid dosage forms
Generally, multi-particulate formulations or multiple unit-dosage forms (pellets,
microtablets and granules) are continuously produced since they permit flexibility in
development. Pellets, being multiple unit-dosage forms, are widely used as they offer
both manufacturing and therapeutic advantages over single-unit solid dosage forms
(DukiC-Ott et al., 2009).
As defined by Ghebre-Sellassie, (1989); pellets are spherical, free-flowing granules with
a narrow size distribution, typically varying between 500 and 1500 µm in size for
pharmaceutical applications. They are formed as a result of a pelletization process which
is an agglomeration process that converts fine powders or granules of bulk drugs and
excipients into small, free-flowing, spherical or semi-spherical units. There are different
techniques applicable for the production of pellets in pharmaceutical industries which
are:
(i) Solution and suspension layering. This process uses conventional coating pan or
fluidized bed with conventional top spray or Wurster bottom spray to apply drug/binder
solution or suspension to solid cores that can be inert materials or granules of the same
drug.
(ii) Dry powder layering. This involves the use of rotor-granulators/rotor tangential
spray fluid-bed to deposit successive layers of dry powder (drug and excipients) on inert
materials with the help of an adhesive solution/binding liquid.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 2
(iii) Direct powder pelletisation. The technique uses high shear mixers and centrifugal
fluid-bed or rotary fluid-bed granulators to apply agglomeration liquid direct to a powder
mixture of a drug and expients followed by pelletization by means of a rotating disc.
A binder can be added as a liquid (wet pelletization) or added as a molten binder before
or during the process (melt pelletization).
(iv) Extrusion-Spheronisation process. This is the most employed technique as it offers
the advantage to incorporate high amounts of active pharmaceutical ingredient, without
producing an excessively large particle of drug-loaded pellets apart from being more
efficient than the other techniques for producing pellets.
Extrusion can be defined as the process of forcing a material through an orifice or die
under controlled conditions thus forming cylinders or strands called extrudates. During
spheronization, these extrudates are broken into small cylinders and consequently
rounded into spheres (pellets). Hence, extrusion/spheronization is a multiple-step process
capable of making uniformly sized spherical particles referred to as pellets and involving
the following sequential steps: (1) dry blending, (2) wet granulation, (3) extrusion, (4)
spheronization, (5) drying, and (6) optional screening (Erkoboni, 2003).
Figure 1.1: Extrusion/spheronization process flow chart with individual processing
variables.
Dry mixing or blending of powders - Equipment type - Mixing time
Wet mixing or granulation of the powders - Equipment type - Solvent or solution - Wet Mixing time - Mixing speed - Granulation liquid level
Extrusion of the wet mass into extrudates
- Extruder type - Screw speed - Screen opening size - Material feeding rate - Extrusion temperature
Spheronization of the extrudates - Spheronizer plate design - Material load - Spheronization speed - Residence time
Drying of the resulting pellets - Equipment type - Drying rate - Drying temperature & time
API EXCIPIENTS
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 3
The end product from each of the steps is shown Figure 2.
Figure 1.2: Product produced by the first four extrusion/spheronization process steps:
(a) Powder from dry mixing, (b) granules from granulation, (c) extrudate from extrusion,
and (d) spheres (pellets) from spheronization.
(http://www.pharmamanufacturing.com/articles/2007/080.html)
1.2. Mechanisms of pellet formation
The transformation from cylinder-shaped extrudate to a sphere occurs in various stages.
Two models have been proposed to describe the mechanism as shown graphically in
Figure 1.3.
Figure 1.3: Pellet-forming mechanisms by spheronization process.
(http://www.pharmamanufacturing.com/articles/2007/080.html )
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 4
One model was proposed by Baert and Remon (1993) who suggested that, the initial
cylindrical particles (Fig.1.3: 1a) are deformed into a bent, rope-shaped particle (Fig.1.3:
1b), then form a dumbbell with a twisted middle (Fig.1.3: 1c). The twisting action causes
the dumbbell to break into two spherical particles, with a flat side having a hollow cavity
(Fig.1.3: 1d). Continued action in the spheronizer causes the particles to round off into
spheres (Fig.1.3: 1e).
The second model proposed by Rowe (1985), describes a transition whereby the
cylindrical particles (Fig.1.3: 2a) are first rounded off into cylindrical particles with
rounded edges (Fig.1.3: 2b), then form dumbbell-shaped particles (Fig.1.3: 2c), ellipsoids
(Fig.1.3: 2d), and finally, spheres (Fig.1.3: 2e) (Erkoboni, 2003).
1.3. Pellet properties
The properties of pellets are listed in Table 1.1 together with different evaluation methods
(Knop and Kleinebudde, 2005; Trivedi et al. 2007).
Table 1.1: Properties of pellets
Properties Requirements Evaluation methods
Particle shape, sphericity Like a sphere, aspect ratio close to 1 (< 1.1) Image analysis
Surface texture/roughness Smooth Image analysis, SEM
Mean particle size Between 0.5mm and 1.5mm Sieve or image analysis
Particle size distribution Narrow Sieve or image analysis
Porosity Low Mercury intrusion
Density High Bulk density determination
Mechanical strength of pellets
High Friability test, crushing strength
Flow properties Very (good) Angle of repose, flow rate estimation
Drug content From very low to more than 95% Dependent on the drug
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 5
Drug release From immediate to controlled release Dissolution tests
1.4. Advantages of pellets
Pellets offer several advantages as multiple unit dosage forms:
• The gastrointestinal transit time, especially the residence time in the stomach, is
more uniform and less sensitive to food ingestion compared to single unit dosage
forms because the small pellets can pass the pylorus even in the closed state. This
leads to reduced variability in drug plasma absorption profiles between subjects
and within the same patient, as a result of even distribution in the GI tract.
• The particles are well dispersed after swallowing and hence the local
concentration of drug is relatively low and irritation of the gastrointestinal mucosa
is therefore less.
• Pellets with a coating for modified release have a lower risk of dose dumping than
coated tablets. That is, immediate release of API from a sustained release single
unit tablet with a defective coating is very much reduced in pellets.
• Pellets with different coatings can be mixed and filled into capsules to enable the
desired dissolution profile to be achieved.
• Pellets with incompatible bioactive drugs can be coated and filled into capsules or
compressed into tablets without the risk of interaction of the substances during
preparation and storage so that can be administered as a single unit dosage form
• They can be formulated as sustained, controlled, or site-specific delivery of the
drug from coated pellets.
• The manufacturing advantages include: - ease of capsule filling because of
improved flow, reduced friability, narrow particle size distribution, ease of
coating and uniform packing (Vervaet, et al., 1995; Knop and Kleinebudde.,
2005; Trivedi et al., 2007).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 6
1.5. Pellet applications
Potential applications of pellets are many and can be categorized into pharmaceutical,
food and agricultural uses as listed below (www.lcicorp.com.) Pellets can be formulated
into controlled release pellets, multi particulate systems, and enteric drugs or developed
as novel drug delivery systems as they permit flexibility in development (Figure 1.4).
Pharmaceutical application of pellets
Figure 1.4: Flow chart for pharmaceutical applications of pellets
Food applications of pellets: Pellets can be formulated for food application purposes as
sugars and sweeteners, catalysts or beverage preservatives.
Agriculture applications of pellets: In agricultural business, pellets can be formulated as
fish foods and also into fertilizer as agricultural chemicals.
Immediate release Controlled /sustained release
Coated pellets Matrix pellets
Compression into tablets
Encapsulation
Compression into tablets
Pellets
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 7
1.6. Process benefits of extrusion/spheronization
Pellets are widely produced by extrusion-spheronization process in pharmaceutical
industries as the technology offers the following benefits: ease of operation and scale-up,
high yield with low wastage, better and narrower particle size distribution, improved
flowability, production of pellets with low friability, production of pellets that are suited
for easy film coating, production of uniformly-sized distributed pellets, production of
wide variety of engineered controlled-release drugs, reproducible packing and low dust
generation.
1.7. Objectives of the study
It is with regard to these resourceful uses that this study identified two areas of research,
based on a food and a pharmaceutical application of pellets as delivery system with the
following objectives:
1. To develop a multiparticulate formulation containing stabilizing agents for improved
colloidal stability in the beer brewing process, via extrusion/spheronization technique
(pellets) and by direct compression method (minitablets) respectively.
2. To develop and characterize fast-disintegrating enteric-coated pellets and minitablets
containing antigen-loaded yeast carriers as vaccine delivery system for oral
administration, via extrusion/spheronization technique (pellets) and by direct
compression method (minitablets) respectively.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 8
References
Baert, L.; and Remon, J.P. (1993). Influence of amount of granulation liquid on the drug
release rate from pellets made by extrusion spheronisation. Int. J. Pharm. 95: 135-141.
DukiC-Ott, A.; Thommes, M.; Remon, J. P.; Kleinebudde, P.; Vervaet, C. (2009).
Production of pellets via extrusion–spheronisation without the incorporation of
microcrystalline cellulose: A critical review. European Journal of Pharmaceutics and
Biopharmaceutics, 71: 38–46.
Erkoboni, K.A. (2003). Extrusion/spheronization, in: I. Ghebre-Sellassie, C. Martin
(Eds.), Pharmaceutical Extrusion Technology, Marcel Dekker Inc., New York and Basel:
277–322.
Ghebre-Sellassie, I. (1989): Pellets: A general overview. In: Pharmaceutical Pelletization
Technology, Ghebre-Sellassie I. (Ed.), Marcel Dekker Inc., New York and Basel: 1-13.
Knop, K.; and Kleinebudde, P. (2005). Pharmaceutical pellets, definition, properties,
production. ExAct Expients and Actives for Pharma, 15: 2-5.
O’Connor R.E., and Schwartz, J.B. (1989). Extrusion and Spheronization Technology. In:
Pharmaceutical Pelletization Technology, Ghebre-Sellassie, I., Martin, C., (Eds.), Marcel
Dekker Inc., New York and Basel. 187-216.
Rowe, R.C. (1985). Spheronisation: A novel pill-making process? Pharm. Int. 6: 119-123.
Trivedi, N.R.; Rajan, M.G.; Johnson, J.R.; and Shukla, A.J. (2007). Pharmaceutical
approaches to preparing pelletized dosage forms using the extrusion-spheronization
process. Critical Reviews™ in Therapeutic Drug Carrier Systems, 24(1): 1- 40.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 9
Vervaet, C.; Baert, L.; and Remon, J.P. (1995). Extrusion-Spheronization, A literature
review. International Journal of Pharmaceutics, 116: 131-146.
www.lcicorp.com (cited 25-4-2010).
http://www.pharmamanufacturing.com/articles/2007/080.html (cited 4-5-2010).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 10
CHAPTER 2:
DEVELOPMENT OF A MULTIPARTICULATE FORMULATION
CONTAINING STABILIZING AGENTS FOR IMPROVED
COLLOIDAL STABILITY IN THE BEER BREWING PROCESS
2.1. Introduction
Colloidal stability of beer can be obtained through the use of polyvinylpolypyrrolidone
(PVPP) or tannic acid. The use of stabilisation products during beer filtration is an
important procedure in brewing processes that determines the shelf life of beer and hence
preserves its stability and quality improvement.
Colloidal instability in beer caused mainly by interactions between haze-forming
polyphenols (proanthocyanidins) and haze-sensitive proteins (polypeptides) during the
shelf life (long term storage) is a persistent problem facing beer brewers and can result in
irreversible non-biological haze and therefore limit the product’s shelf life.
2.2. Main sources of haze precursors
Raw materials and process stages are the two area sources of these haze-forming
precursors in packaged beer. Several steps are involved in the brewing process which
include, malting, milling, mashing, lautering, boiling, fermenting, conditioning, filtering
and packaging.
Polypeptides which are responsible for haze-active proteins are high molecular weight
compounds originating mainly from barley and are rich in amino acids proline and
glutamic acid, and heavily glycosylated with glucose accounting for 3-7% of total beer
protein (Leiper et al,. 2003). These polypeptides, known as sensitive proteins, will
precipitate with tannic acid, which provides a means to determine their levels in beer.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 11
Proline sites in the haze-active polypeptides bind to the gallotannins so that haze-forming
proteins are selectively adsorbed.
Polyphenols in beer originate from barley and hops. Their structure is based on phenol
(monohydroxylated benzene) and the term covers all molecules with two or more phenol
rings and beer contains approximately 100-300mg/L polyphenol (McMurrough and
O’Rourke, 1997). These polyphenols can be divided into derivatives of hydrobenzoic and
hydroxycinnamic acids and flavanols and their derivatives. Flavanols and their
derivatives account for 10% of total beer polyphenols and contain the species related to
colloidal instability (Leiper et al., 2005). Flavanols found in beer are catechin,
epicatechin, gallocatechin and epigallocatechin (Figure 2. 1), which all exist as
monomers but are more commonly joined to form flavonoids as dimers, trimers or larger
polymers. Flavonoids found in beer consist of monomers, dimers and a few trimers at a
level of approximately 15mg/L. Two dimers that have been associated with haze
formation are procyanidin B3 (catechin-catechin) and prodelphinidin B3 (gallocatechin-
catechin) (Figure 2. 1). These are known as proanthocyanidins and come from malt and
hops accounting for 3.3% of total beer polyphenols (McMurrough and O’Rourke, 1997).
Figure 2.1: Structures of the main flavanol monomers and dimers (Leiper et al., 2005).
Polyphenols that are polymerized and that can precipitate with proteins are called tannins.
Both barley and hops contain condensed tannins or proanthocyanidins and these represent
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 12
the essential substances responsible for colloidal instability, i.e. they are the haze-active
polyphenols (Hannes et al., 2007). Polypeptides and polyphenols combine to produce
visible haze that reduces beer’s physical shelf life. The haze proteins have regions rich in
the amino acid proline, to which the polyphenols attach. Reducing the levels of one or
both of the haze precursors using suitable stabilizing treatments will extend physical
stability. The instability can manifest as chill haze (haze formed at a temperature lower
than 4 degrees Celsius) or as permanent haze (haze formed at room temperature) (Leiper
et al., 2005; Rehmanji et al., 2005; Hannes et al., 2007). Since the two major components
of colloidal haze are the protein and polyphenol fractions, their reduction in beer prior to
packaging is the obvious target.
2.3. Strategies to minimize haze precursors formation
Colloidal stabilisation of beer by selective removal of either one or both of the haze
precursors with suitable stabilizing treatments is the key approach to extend the shelf life.
Most widely employed agents in current use for stabilization are PVPP for polyphenol
haze precursor reduction and gallotannin for protein haze precursor reduction (Rehmanji
et al., 2005). The technique is done by first adding gallotannin powder that adsorbs haze-
active proteins by hydrogen bonding followed by addition of PVPP micronized powder
that adsorbs tannoids or proanthocyanidins and haze polyphenols through H-bonding
between the proton donor (hydroxyl groups) from polyphenols and carbonyl groups from
PVPP (Figure 2.2).
Figure 2.2: Structure of Polyvinylpyrrolidone/Polyvinylpolypyrrolidone (PVPP).
http://en.wikipedia.org/wiki/Crospovidone
The use of PVPP results in a decrease of the amount of haze-active proanthocyanidins
(and monomeric catechins) without a negative influence on beer foam stability (Hannes
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 13
et al., 2007). The addition of these compounds is done during beer filtration for a
specified contact time period.
Other options for achieving good colloidal stability that are being investigated include a
novel upstream beer colloidal stabilization employed in the brewhouse toward the end of
wort boiling. Upstream beer stabilization technique is easier to employ because the
stabilization product is added directly to the boiling wort without the need for specialized
equipment, such as slurry tanks and dosing units. This has the advantage of improving
wort clarity as well as increasing the shelf life of the beer and thus provides the most
effective colloidal stabilization (Rehmanji et al., 2005). Wort is the extracted liquid from
the mashing process and contains sugars (mainly maltose & maltotriose from starch in
malted barley, and other compounds) that will be fermented by the brewing yeast to
produce alcohol.
Recent studies have indicated that the combined use of PVPP and tannic acid (Figure 2.3)
at the end of wort boiling has a positive impact on colloidal and flavour stability. The use
of tannic acid, one minute later followed by PVPP, ensures good shelf life of the beer. A
combined product which ensures that the tannic acid come firstly in contact with the
boiling wort and short time later the PVPP, is a straightforward application type for the
brewery sector (Hannes et al., 2007).
Figure 2.3: Structure of Tannic acid (http://www.chemblink.com/products/1401-55-4)
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 14
2.4. Aim of the study
Therefore, the aim of this study was to develop a combined product (PVPP with
gallotannin) in form of multiparticulates (pellets or minitablets) that would provide
colloidal stabilization as a single product when added before the end of wort boiling and
thus introduce the concept of a balanced reduction of both major classes of haze
precursors—polyphenols and proteins.
PVPP and gallotannin are incompatible and therefore, a primary coating polymer or
layering solution should be sprayed first on the PVPP pellets/minitablets that will
separate the two compounds before gallotannin is sprayed in order to develop as single
product which ensures that the tannic acid comes first into contact with the boiling wort
and short time later the PVPP.
During this study, various PVPP-based pellets with varying concentrations were
formulated and tested for disintegration in order to comply with the required contact time
of the adsorbent in tank or at the filter point (7 minutes exposure of PVPP in contact with
boiling wort) before combining with gallotannin by fluid bed coating to finally develop a
combined product. Minitablets (5mm diameter) were also formulated for the same study.
2.5. Materials and Methods
2.5.1. Production of PVPP-based pellets
Pellets were prepared via the extrusion-spheronization technique. Each batch (batch size:
300g) contained polyvinylpolypyrrolidone (Polyclar® 10) (60% - 95%w/w) and
additional excipients at varying concentrations. The additional excipients were added in
various amounts (between 5% - 40%w/w) so that the prepared pellets disintegrate fast
enough (within 7 minutes in boiling simulated wort solution).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 15
The following materials were used during preliminary tests: PVPP (Polyclar® 10; 40 µm
and 100µm particle size) supplied by ISP (Switzerland) A.G. Other materials
incorporated at various amount included: Unipure EX® starch, potato starch (C*Gel
3002), sodium bicarbonate, glucose monohydrate, sodium starch glycolate (Explotab®),
microcrystalline cellulose (Avicel® PH 102), sodium alginate, silicon dioxide hydrate,
kappa carrageenan, lambda carrageenan, iota carrageenan, microcrystalline cellulose &
sodium carboxymethylcellulose (Avicel®RC 581), polyvinyl pyrrolidone (Kollidon
30®), and hydroxypropylmethylcellulose. Demineralised water was used as granulating
liquid.
In all prepared formulations, powdered PVPP (Polyclar® 10) with corresponding
ingredients (batch size: 300g) were dry mixed for 15 minutes using a planetary mixer
(Kenwood Chief, Hampshire, UK) with a K-shaped mixing arm to obtain a uniform
powder blend. Specified amount of granulating liquid (demineralised water) was added at
once to the powder mixture in the same mixer and blended for 5 minutes to form a wet
granulated mass ready for extrusion. The wet mass was extruded axially at a speed of 50
rpm using a single screw extruder (Dome extruder, Lab model DG-L1, Fuji Paudal,
Tokyo, Japan) fitted with dome-shaped screen of 1mm diameter perforations and 1.2mm
thickness. The formed extrudates were spheronized for 5 minutes in a spheronizer
(Caleva Model 15, Caleva, Sturminster Newton, Dorset, UK) with a cross-hatched
friction plate at a speed of 850 rpm to form pellets followed by drying in an oven at 40°C
for 24hrs.
2.5.2. Production of PVPP minitablets
Minitablets of 5mm diameter containing polyvinylpolypyrrolidone (Polyclar® 10), of
40µm and 100µm particle sizes, and Lambda Carrageenan were also prepared by direct
compression of uniform granules.
PVPP and Lambda carrageenan were dry mixed (batch size 100g) for 15 minutes in a
Turbula® mixer (model T2A, W.A. Bachofen, Basel, Switzerland) to obtain a uniform
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 16
powder mixture. Demineralized water was added to the powder blend and mixed for 5
minutes in a mortar and pestle to form wet granules that were sieved through 1480µm
mesh and left to dry for 24 hours at ambient temperature condition.
Dried granules (50g) were sieved through 750µm mesh followed by addition of
magnesium stearate. After mixing in a turbula mixer for 15 minutes, the granules were
tableted at 25 - 60 kgf (kilogram-force, measured by digital indicator, AD-4532A
connected to the tablet press) using a single punch eccentric tablet press (type EKO,
Korsch, Berlin, Germany).
Table 2.1a: Composition of PVPP-based granules
Ingredient Quantity (g)
Polyvinylpolypyrrolidone (Polyclar® 10) 80.0
Lambda Carrageenan 20.0
Demineralized water 90.0
Table 2.1b: Composition of PVPP minitablets
Ingredient Quantity (g)
PVPP-Lambda Carrageenan dried granules (750 µm) 50.0
Magnesium stearate 0.25
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 17
2.5.3. Preparation of simulated wort solution
300ml of 12% w/v sucrose solution was prepared and its pH adjusted to 5.0-5.2 by 1%
v/v lactic acid and used as a simulated wort boiling solution to determine the
disintegration time for the prepared pellets. A pH between 5.0-5.2 is desired in order to
obtain clear worts (Withouck et al., 2010).
2.5.4. Pellet disintegration test
100mg of each prepared batch of pellets was dispersed in 300ml of 12% w/v boiling
sucrose solution at pH 5.0-5.2 (Figure 2.4a), which was used as a simulated wort boiling
solution in this study. Dispersed pellets were observed for complete disintegration of 7
minutes since this is the time allowed for added PVPP powder to adsorb polyphenols in
wort boiling. Similar experiment condition was set for PVPP powder (100mg dispersed
in 300ml of 12% sucrose boiling solution, pH 5.0-5.2) (Figure 2.4b) to form dispersion
that served as a physical control reference for assessing the complete disintegration of
pellets.
Figure 2.4a Figure 2.4b
Figure 2.4a: Disintegration test for 85%PVPP +15% Explotab pellets in 12% sucrose
solution.
Figure 2.4b: PVPP powder in 12% boiling sucrose solution.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 18
2.5.5. Tablet disintegration test
A similar disintegration test was carried out for the prepared minitablets as explained (in
section 2.5.4.) above by placing one tablet as a representative of the batch in 300ml of
12% w/v boiling sucrose solution at pH 5.0-5.2 and record the time taken for
disintegration. The test was repeated three times.
2.5.6. Tablet hardness test
A sample of minitablets (n=20) (compressed at 20 - 30kgf) was tested for hardness test
using a tablet hardness tester, (Type PTB 311, Pharma Test, Hainburg, Germany) and the
method as described in European Pharmacopoeia, 6th edition. The test jaws constant
speed was 1.00 mm/sec. Results were expressed as the mean, minimum and maximum
values of the forces measured, all expressed in Newtons representing twenty
determinations.
2.5.7. Tablet friability test
Minitablets (n=20) from the same batch (compressed at 20 - 30kgf) were tested for
friability as described in European Pharmacopoeia, 6th edition monograph for friability of
uncoated tablets using a friabilator, (Type PTF, Pharma Test, Hainburg, Germany).
A sample of twenty minitablets (Ws) was weighed and placed on a sieve 1000 µm size to
remove any loose dust with the aid of air pressure before being placed in a friability drum
and fitted to a friabilator. The sample was subjected to roll and fall by rotating the drum
100 times and then followed by removal of any loose dust generated from the minitablets
during the test. The tablets were weighed again to determine their final weight (Wf). The
friability was expressed as the loss of mass and was calculated as a percentage of the
initial mass using the following equation:
( )( )
100% ∗
−=
Ws
WfWsFriability (Equation 2.1)
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 19
2.6. Results and discussion
2.6.1. Pellet disintegration test
PVPP-based pellets that were prepared and tested for disintegration in simulated wort
boiling, 12% sucrose soln, pH 5.0-5.2 are presented in Table 2.1. During extrusion
process majority of the prepared batches produced extrudates that were plastic and rigid
enough to withstand spheronization process and spheronized well to produce spherical
pellets, but could not disintegrate completely in 7 minutes which is the recommended
contact time. Contact time is the time that the stabilization product is in contact with the
wort at the end of boiling (Hannes et al., 2007).
On the other hand, some batches produced plastic and rigid extrudates, but could not
form spherical pellets during spheronization and hence ended in forming short rodlike
particles. Other materials did not spheronize at all probably lacking strong bonding
forces that contribute to pellet formation (Ghebre-Sellassie, 1989b).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 20
Table 2.1: Summary of prepared PVPP-based pellets tested for disintegration test in 300 ml, 12% boiling sucrose solution.
Batch no Preparations name Extrudates quality Pellets shape Observation/results
001 95% PVPP (40µm) + 5% unipure®ex starch
rigid enough spherical Slightly disintegrated
002 90% PVPP(40µm) + 10% MCC rigid enough spherical Slightly disintegrated
003 90% PVPP(40µm) + 10% C*Gel 3002 rigid enough spherical Slightly disintegrated
004 90% PVPP(40µm) + 5% unipure®ex starch + 5% Sodium bicarbonate
rigid enough spherical Slightly disintegrated
005 90% PVPP(40µm) + 10% Sodium bicarbonate
rigid enough spherical Slightly disintegrated
006 100% PVPP(40µm) rigid enough spherical Slightly disintegrated
007 80% PVPP(40µm) + 20% Glucose monohydrate
rigid enough spherical Slightly disintegrated
008 70% PVPP(40µm) + 30% Glucose monohydrate
rigid enough spherical Slightly disintegrated
009 85% PVPP(40µm) + 15% Explotab® rigid enough
spherical Disintegrated completely
010 85% PVPP(40µm) + 5% Glucose monohydrate + 5% Explotab® + 5% Sodium bicarbonate
rigid enough spherical Slightly disintegrated
011 100% PVPP(40µm) (extruded at 10rpm) rigid enough spherical Slightly disintegrated
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 21
012 80% PVPP(40µm) + 20% Unipure®ex starch
rigid enough
spherical Slightly disintegrated
013 80% PVPP(40µm) + 20% Sodium Alginate
rigid enough short rodlike particles Slightly disintegrated
014 80% PVPP(40µm) + 20% Silicon dioxide hydrate
rigid enough
spherical Slightly disintegrated
015 80% PVPP(40µm) + 20% Kappa Carrageenan
rigid enough spherical Slightly disintegrated
016
80% PVPP(40µm) + 20% Lambda Carrageenan
rigid enough
short rodlike particles
Disintegrated completely
017 85% PVPP(40µm) + 15% Lambda Carrageenan
rigid enough
short rodlike particles Slightly disintegrated
018 90% PVPP(40µm) + 10% Lambda Carrageenan
rigid enough
short rodlike particles Slightly disintegrated
019 95% PVPP(40µm) + 5% Lambda Carrageenan
rigid enough
short rodlike particles Slightly disintegrated
020 100% PVPP (100µm) short soft extrudates
Did not spheronize
Not tested
021 80% PVPP(100µm) + 20% Lambda carrageenan
rigid enough
short rodlike particles Disintegrated completely
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 22
022 75% PVPP (100µm) + 20% Unipure®ex starch + 5% HPMC
rigid enough
spherical Slightly disintegrated
023 70% PVPP(100µm) + 20% Lambda carrageenan + 10% Unipure®ex starch
rigid enough
short rodlike particles Disintegrated completely
024 80% PVPP (100µm) + 10% Kollidon 30® + 10% HPMC
rigid enough
spherical Slightly disintegrated
027 70% PVPP (40µm) + 20% Lambda carrageenan+10% Kappa carrageenan
rigid enough
short rodlike particles Disintegrated completely
028 80% PVPP (40µm) + 20% Iota carrageenan
rigid enough
short rodlike particles Slightly disintegrated
029 80% PVPP (40µm) + 20% Lambda carrageenan
rigid enough
short rodlike particles Disintegrated completely
030 90% PVPP(100µm) + 10% Kappa carrageenan
rigid enough
spherical Slightly disintegrated
031 60% PVPP (100µm)+ 20% Lambda carrageenan + 20% MCC (Avicel®PH 102)
rigid enough
short rodlike particles Disintegrated completely
032 80% PVPP (100µm)+20% MCC & Sodium carboxymethylcellulose (Avicel®RC 581)
rigid enough
spherical Disintegrated completely
033 80% PVPP (40µm)+20% MCC & Sodium carboxymethylcellulose (Avicel®RC 581)
rigid enough spherical Slightly disintegrated
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 23
034
100% Polyclar® Brewbrite (Pvpp & Kappa Carrageenan)
rigid enough
spherical
Slightly disintegrated
035 100% Polyclar® Plus 730
(Pvpp + amorphous silica gel)
No extrudates formed.
did not spheronize Not tested
036 50% PVPP (100µm) + 50% Unipure®ex starch
Too short and soft. did not spheronize Not tested
037 50% PVPP (40µm) + 50% Unipure®ex starch
rigid enough spherical
Slightly disintegrated
038 70% PVPP (40µm) + 30% Unipure®ex starch
rigid enough
spherical Slightly disintegrated
039 68% PVPP (40µm) + 30% Unipure®ex starch + 2% Lambda Carrageenan
rigid enough
short rodlike particles Slightly disintegrated
040 80% PVPP (40µm) + 10% Kappa Carrageenan + 10% Avicel®RC 581.
rigid enough spherical Slightly disintegrated
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 24
PVPP-based pellets that were preliminarily tested for disintegration in simulated wort
boiling, 12% sucrose soln, pH 5.0-5.2) and disintegrated completely in 7 minutes and
hence comply with the contact time required for the PVPP powder to adsorb
polyphenols at the end of wort boiling in tank or at the filter point are presented in Table
2.2.
Table 2.2: Disintegration test results for PVPP-based pellets in simulated wort (12%
sucrose solution, pH 5.0-5.2).
Batch no Preparations name Disintegration time
009 85% PVPP (40µm) + 15% Sodium starch
glycolate with Sodium Carboxymethyl Starch
(Explotab®)
7 minutes
016 80% PVPP (40µm) + 20% Lambda Carrageenan 7 minutes
021 80% PVPP (100µm) + 20% Lambda Carrageenan 7 minutes
023 70% PVPP (100µm) + 20% Lambda Carrageenan
+ 10% Unipure® ex starch
7 minutes
027 70% PVPP (40µm) + 20% Lambda Carrageenan +
10% Kappa Carrageenan
7 minutes
031 60% PVPP (100µm) + 20% Lambda Carrageenan
+ 20% Microcrystalline cellulose (Avicel® PH
102)
7 minutes
032 80% PVPP (100µm) + 20% Microcrystalline
cellulose & Sodium carboxymethylcellulose
(Avicel®RC 581)
7 minutes
Preparations, batch no 009 and 032 were tested in real wort (malted barley and hops)
laboratory scale trials and their results are presented in Table 2.3. The results are
presented in percentage concentration using blank (100% wort without PVPP treatment)
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 25
as a standard reference. The tested real wort was treated with PVPP-based pellets at 10
g/hl for contact time of 7 minutes.
During wort treatment with PVPP-based products (powder, pellets or tablets), attention is
paid to the reduction of proanthocyanidins content, the less proanthocyanidins, the better.
The polyphenol concentration gives an overview of the total polyphenols (partly
proanthocyanidins and flavonoids) present in the wort. The sensitive proteins are
removed by addition of gallotannin as mentioned earlier. PVPP is considered to be
effective when proanthocyanidins level is reduced between 30 - 40% with reference to
the blank.
Table 2.3: Effectiveness of PVPP-based pellets in reducing proanthocyanidins
concentration in real wort laboratory scale trials
Flavonoids Polyphenols Proanthocyanidins Sensitive proteins
Batch 009 95% 95% 88% 126%
Batch 032 97% 98% 96% 106%
According to the results obtained from the wort laboratory scale test shown in Table 2.3,
there was a reduction in the level of proanthocyanidins (88% and 96%) when the wort
was treated with 85% PVPP + 15% Sodium starch glycolate (Explotab®) and 80%
PVPP + 20% Microcrystalline cellulose & Sodium carboxymethylcellulose (Avicel®RC
581) pellets, respectively in relation to blank. However, the reduction was not sufficient
enough and therefore, it was decided to develop a combination of minitablets containing
PVPP and lambda-carrageenan. PVPP + lambda-carrageenan was the only combination
that disintegrated completely in 7 min, but it was not possible to make pellets of this
combination as they formed short rodlike particles, and hence not spheronisable (Table
2.1, batch no. 021).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 26
It is worthwhile to mention that carrageenan, which is a natural product extracted from
red marine algae, has a long history of use in brewing as a wort clarifier. It is effective in
reducing haze-precursors in wort and these include protein, polyphenol, polysaccharide
and other materials (ISP: Polyclar® BrewbriteTM, 2004, version Jan-05). Therefore, its
inclusion in these preparations also reduces the formation of protein-polyphenol
complexes.
2.6.2. Tablet disintegration test
Minitablets (80% PVPP (40µm) + 20% Lambda Carrageenan, and 80% PVPP (100µm)
+ 20% Lambda Carrageenan) disintegrated in 7 minutes when preliminarily tested in
simulated wort (12% sucrose solution) as described earlier and hence achieved the
disintegration test to comply with the time required when PVPP is dispersed in wort at
the end of boiling.
The prepared minitablets were further tested in real wort produced at laboratory scale and
the results are presented in Table 2.4.
Table 2.4: Effectiveness of PVPP-based minitablets in reducing proanthocyanidins
concentration in real wort laboratory scale trials
Flavonoids Polyphenols Proanthocyanidins Sensitive proteins
TMT1 101% 99% 68% 128%
The tested real wort was treated with PVPP-based tablets at 15g/hl for contact time of 7
minutes.
Proanthocyanidins level was reduced to 68% (a reduction of 32% in relation to blank ,
which is 100% wort without PVPP addition as reference standard) when minitablets
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 27
containing 80% PVPP and 20% Lambda Carrageenan were used. This indicated that
PVPP was effective.
2.6.3. Tablet hardness test
The hardness of a tablet refers to a measure of the crushing/breaking force under
diametrical loading. The mean, minimum and maximum measured forces for the tested
minitablets (n=20; compressed at 20-30kgf) were 23N, 18N and 33N respectively. Since
the hardness of a tablet affects its disintegration time, the harder the tablet, the longer it
takes to disintegrate. The obtained values are considered acceptable in relation to this
study since the minitablets disintegrated in 7 minutes as required.
2.6.4. Tablet friability test
Friability refers to the tendency for the tablets to form dusts or to break-up/off when
subjected to abrasion forces. It is the ability of the compressed tablet to avoid fracture and
breaking apart during transport.
A prepared combination of 80% PVPP and 20% Lambda Carrageenan minitablets
(compression force, 20-30kgf) had a high mechanical strength, since friability values of
less than 1% were obtained (friability = 0.73%). The pharmacopeial limits consider a
maximum loss of 1 percent of the mass of the tablets tested to be acceptable. This low
friability indicates the tablet’s ability to withstand the shear forces when subjected to
mechanical shock or attrition.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 28
2.7. Conclusion
Preliminary results indicate that the composition of the minitablets containing 80% PVPP
and 20% lambda-carrageenan has managed to reduce the formation of polyphenols in
worts on laboratory scale. These results will pave the way to the next step of developing a
combined product that will be applied in brewing trials and implemented in several
industrial “case” studies in order to achieve colloidal stability by reducing these protein-
polyphenol complexes. By using fluid bed coating technique, the PVPP minitablets will
first be coated with maltodextrin solution and finally, coated with gallotannin.
Prepared PVPP-based pellets, though tested, could not reduce significantly the amount of
haze-forming precursors after the required disintegration time in wort boiling.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 29
References
Hannes, W.; Annick, B.; Patrick, D.; Gerda, D.; Gert, De R.; and Roger, M. (2007).
Upstream beer colloidal stabilization during wort boiling by addition of a
Polyvinylpolypyrrolidone or a gallotannin, Cerevisia, 32, (4).
Ghebre-Sellassie I., Mechanism of pellet formation and growth. In: Pharmaceutical
Pelletization Technology, Ghebre-Sellassie I. (Ed.), Marcel Dekker Inc., New York and
Basel (1989b) 123-143.
ISP: Polyclar® BrewbriteTM. Wort Clarifier & Beer Stabilizer. International Specialty
Products, 2004, version Jan-05: 1-4.
Leiper, K.A.; Stewart, G.G.; McKeown, I.P.; Nock, T.; and Thompson, M.J. (2005).
Optimising beer Stabilisation by the Selective Removal of Tannoids and Sensitive
Proteins. Journal of the Institute of Brewing, 111(2): 118-127.
Leiper, K.A.; Stewart, G.G.; McKeown, I.P. (2003). Beer polypeptides and silica gel.
Part I. Polypeptides involved in haze formation. Journal of the Institute of Brewing, 109:
57-72.
Rehmanji, M.; Gopal, C.; and Mola, A. (2005). Beer Stabilization Technology- Clearly a
Matter of Choice. Technical quarterly of the Master Brewers Association of the
Americas, 42(4): 332-338.
McMurrough, I.; and O’Rourke, T. (1997). New insight into the mechanism of achieving
colloidal stability. Technical Quarterly of the Master Brewers Association of the
Americas, 34: 271-277.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 30
Withouck, H.; Boeykens, A.; Jaskula, B.; Goiris, K.; De Rouck, G.; Hugelier, C.;
Aerts. G. (2010). Upstream Beer Stabilisation during Wort Boiling by Addition of
Gallotannins and/or PVPP. BrewingScience January / February 2010 (Vol. 63):14-22
http://www.chemblink.com/products/1401-55-4 (cited on 6-5-2010)
http://www.thoroughbrews.com/pb/wp_b120e579/wp_b120e579.html (cited on 6-5-
2010)
http://en.wikipedia.org/wiki/Brewing (cited on 6-5-2010)
http://en.wikipedia.org/wiki/Crospovidone (cited on 6-5-2010)
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 31
CHAPTER 3:
FORMULATION OF ENTERIC COATED PELLETS AND
MINITABLETS CONTAINING ANTIGEN-LOADED YEAST
CARRIERS
3.1: Vaccination
Vaccination was introduced by Jenner (1749 –1823) and Pasteur (1822 – 1895) in the late
18th and 19th centuries using attenuated or killed microorganisms to induce protective
immunity against several pathogens (Geison, 1978; Stewart and Devlin, 2006). The
methods are still applicable to date and have led to the control of infectious diseases such
smallpox, diphtheria, tetanus, pertussis, polio and rabies worldwide (WHO/IVB/2009).
A vaccine is a biological preparation typically containing an agent that resembles a
disease-causing microorganism, and is often made from weakened or killed forms of the
microbe or its toxins to be administered to humans or animals in order to improve
immunity to a particular disease. All vaccines work by presenting a foreign antigen to the
immune system in order to evoke an immune response. The agent stimulates the body’s
immune system to recognize the agent as foreign, destroy it, and remember it, so that the
immune system can more easily recognize and destroy any of these microorganisms that
it will later encounter. The pathogen is made harmless without loss of antigenicity and
therefore allows an individual to acquire specific immunity to an infectious agent without
having to suffer an initial infection. When administered, it induces antibodies against the
pathogens or toxins and also induces a cellular response and thus generates a strong
immune response to provide long term protection against infection.
Despite the efficacy of killed and attenuated vaccines, there is still a concern over their
safety. Killed bacterial or viral vaccines often have residual toxicity following
inactivation and might contain toxic components, such as lipopolysaccharide (LPS).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 32
Attenuated live vaccines may induce disease via reversion or mutation to an infectious
agent to people with an impaired immune system (Perrie, 2006). This has changed the
focus on vaccine development to safer, although often less immunogenic, subunit
vaccines composed of purified antigenic components of the microorganism. Attenuated
live vaccines generally possess a natural adjuvant capability that is built on the ability of
our immune system to recognize many potential dangerous microbes and thus contribute
to the control of infectious diseases to the immunized population (Aguilar and Rodriguez,
2007). Unlike attenuated live vaccines, highly purified recombinant molecules or
subunits of pathogens lacks inherent immunostimulatory property of the pathogens, and
thus do not often elicit strong immune responses to reach their full potential.
Consequently, the development of subunit vaccines composed of purified antigenic
components or recombinant molecules of the microorganism would require the addition
of an adjuvant in order to be effective (Petrovsky and Aguilar., 2004). The term
“adjuvant” is derived from the Latin word adjuvare which means to help. Any material
that helps the antigens or increases the humoral and/or cellular immune response to an
antigen is referred to as an immunologic adjuvant. Adjuvants have been in use to
augment the immune response to antigens (Gupta and Siber., 1995).
The only universally licensed adjuvant for human vaccine is an Aluminium-based
mineral salt (alum) (Gupta and Siber, 1995; Aguilar and Rodriguez., 2007) which is
widely used in Diphteria, Tetanus, Pertussis and Hepatitis A and B vaccines (Vogel et al.,
1995). Alum’s success as adjuvant is associated with enhanced antibody responses and its
mechanism has recently been revealed to act through activation of the Nacht Domain-,
Leucine-Rich Repeat (LRR) -, and PYD-Containing Protein 3, (NALP3) inflammasome
by induction of uric acid (Kool, 2008). Although alum enhances antibody responses to
parenterally delivered vaccines, it is not effective at inducing cellular immunity and is not
suitable for mucosal immunization (Moore et al., 1995). It is also faced with cumulative
aluminium toxicity and induction of allergic immunoglobulin-E (IgE) antibody responses
(Aggerbeck et al., 1995; Gupta, 1998).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 33
Ideally, adjuvants are required to be strong enough to stimulate B- and T-cell immunity
while avoiding the excess activation of innate immune system and inflammatory cytokine
production that mediates adjuvant reactions and side effects to a patient (Petrovsky,
2008). Adjuvant side effects can be grouped into local and systemic reactions. Local side
effects range from increased injection site pain, inflammation and swelling, granulomas,
sterile abscess formation, lymphadenopathy and ulceration. Systemic vaccine reactions
may include nausea, fever, adjuvant arthritis, uveitis, eosinophilia, allergic reactions,
organ-specific toxicity, anaphylaxis or immunotoxicity mediated by liberation of
cytokines, immunosuppression or induction of autoimmune diseases (Waters et al. 1986;
Aguilar and Rodriguez, 2007).
A new vaccine adjuvant can be approved by the regulatory body if it clearly demonstrates
its safety, tolerability and acceptability that outweigh any increased reactions. Hence, a
major challenge in adjuvant development is how to achieve a potent adjuvant effect while
avoiding reactions or toxicity (Gupta and Siber, 1995). Thus, the need to develop novel
adjuvants against pathogens that have not been managed (e.g. Malaria and HIV/AIDS) by
the traditional vaccination strategies and to overcome the limitations of the available
licensed adjuvants for human vaccines is vital (Gupta, 1998; Harandi et al., 2010).
Despite that, aluminum salt-based adjuvants continue to be the only adjuvants licensed
for human vaccines delivery system, natural and synthetic compounds have also been
identified that have adjuvant activity. The goal at the development of new vaccine
delivery systems is to identify optimal methods for presenting target antigens to the
immune system that will elicit immune responses appropriate for protection against, or
treatment of, a specific disease. A number of novel adjuvants, which may be used to
supplement or replace alum in human vaccines, have been under development and in
preclinical evaluation for several decades (Gupta and Siber, 1995; Ryan et al., 2001;
Moingeon et al., 2002; Aguilar & Rodriguez, 2007).Despite the enormous number of
candidates tested, alum has not been replaced in human vaccines because the potency of
these candidates has been regularly associated with increased toxicity, and this has barred
their use, particularly in prophylactic vaccines where safety issues are vital. Therefore,
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 34
there is still a major need for the development of safe and efficacious adjuvant that is
capable of boosting cellular and humoral immunity (Gupta, 1998; Petrovsky, 2006).
Hence, the aim of this study was to develop enteric coated pellets and minitablets
formulation containing antigen-loaded yeast carriers/shells as delivery system for oral
vaccination via extrusion/spheronization and direct compression methods respectively.
The yeast shells to be used as carriers of antigen were extracted from Baker’s yeast
(Saccharomyces cerevisiae) cell walls that contain yeast-beta glucan as the main
component.
3.2: Structure and classification of glucans.
Glucans are natural polysaccharides comprised of glucose monomers linked by
glycosidic bonds (Figure 3.1). They are classified according to the type of intra-chain
linkage of the polymer into alpha (α-) and beta (β) - glycosidic bonds, depending on
whether the substituent groups on the carbons flanking the ring oxygen are pointing in the
same or opposite directions.
Figure 3.1: General structure of glucan (Novak and Vetvicka, 2008).
An alpha glucan (α-glucan) has α-glycosidic bond which emanates below the plane of the
sugar, whereas the hydroxyl (or other substituent group) on the other carbon points above
the plane (opposite configuration) (Figure 3.2a). The beta glucan (β-glucan) has a β-
glycosidic bond which emanates above that plane (Figure 3.2b).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 35
Figure 3.2: (a) alpha-D-glycosidic linkages Figure 3.2: (b) β-(1,3)- & β-(1,6)-D-
glycosidic linkages
(http://en.wikipedia.org/wiki/Alpha_glucan).
(http://en.wikipedia.org/wiki/Beta_glucan).
Among the common alpha-D-glucans include, α-(1,6)-D-glucan (Dextran); α-(1,4)-D-
glucan (Amylose-mostly linear polymer; Figure 3.3a) and Amylopectin which is α-(1,4)-
D-glucan (highly branched polymer of glucose, Figure 3.3b).
Figure 3.3a: Amylose, α-(1,4)-D- glucan. Figure 3.3b: Amylopectin α-(1,4)-D-
glucan
(http://en.wikipedia.org/wiki/Starch).
Naturally, β-Glucans occur as glucose polymers, consisting of a backbone of β-(1,3)-
linked by β-D- glucopyranosyl units with β-(1,6)-linked side chains of varying
distribution and length (Figure 3.5). These polysaccharides are commonly named “β-
glucans” although they are chemically heterogeneous, and the one referred in this work,
are non-cellulosic polymers of β-D-glucose, with glycosidic bonds in position β-(1,3).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 36
3.3 Sources of glucans
Glucans are found naturally as highly conserved cell wall structural components in
mushrooms (i.e. Maitake, Reishi, and Shiitake –Figure 3.4), yeast, bacteria,
seaweed/algae and wheat (i.e. cereal grains of oats and barley). The beta-linked glucans,
predominantly, β-(1,3)-D-glucans are found in fungi (Wasser, 2002; Brown and Gordon,
2003).
Figure 3.4: Edible mushrooms, e.g. shiitake (Lentinula edodes), a source of natural beta-
glucans. (http://en.wikipedia.org/wiki/Beta_glucan)
The cell wall of the common baker’s or brewer’s yeast, Saccharomyces cerevisiae (S.
cerevisiae) is one of the readily available sources of β-(1,3)-D-glucan. S. cerevisiae,
being yeasts (unicellular fungi), are commonly used in bakery and beer industries for
baking and beer fermentation respectively for many years (Otero et al., 1996). Other
fungal and bacterial sources of β-glucans with immunomodulatory effects are listed in
Table 3.1.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 37
Table 3.1.Common used biologically active β-glucans from fungal and bacterial sources
(Novak and Vetvicka, 2008).
Name Source
Curdlan Bacteria; Alcaligenes faecalis
Laminaran Seaweed; Laminaria sp
Pachymaran Poria cocos
Lentinan Lentinus edodes
Pleuran (HA-glucan) Pleurotus ostreatus
Schizophyllan Schizophyllum commune
Sclerotinan (SSG) Sclerotinia sclerotiorum
Scleroglucan Sclerotium glucanicum, S. rolfsii
Grifolan Grifola frondosa
Yeast glucan Saccharomyces cerevisiae
3.4 Composition of fungal cell walls
The fungal cell walls are composed mostly of β-(1,3)-glucans and mannoproteins with
branched β-(1,6)-glucan that links the components of the inner and outer walls. Other
minor component includes chitin, which contributes to the insolubility of the fibers. The
β-(1,3)-glucan-chitin complex is the major constituent of the inner wall while the outer
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 38
surface of the wall are mannoproteins. Other components found and covalently linked to
a great extent include glycoprotein, lipids, and carbohydrates such as mannan (Lipke and
Ovalle., 1998; Klis et al., 2002; Chan et al., 2009) (Figure 3.5).
Figure 3.5: Components of the fungal cell wall and the structure of β-D-glucan showing
a linear backbone of β-(1,3)-linked glucose with β-(1,6)-linked side branches (Chan et al.,
2009).
The first investigation and preparation of yeast-glucan from the cell wall of baker’s yeast
was reported by Pillemer and Ecker (1941), who revealed the presence of a glucan-
enriched product called Zymosan in the cell wall. Other studies that supported the
presence of zymosan in S. cerevisiae were done by Bacon and Farmer, (1968) and
Manners et al., (1973). They found that, the major component (about 85 %) is a branched
β-(1,3)-glucan of high molecular weight (about 240,000 or 240kDa) containing 3% of β-
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 39
(1,6)-glucosidic interchain linkages and the minor component is a branched β-(1,6)-
glucan. Earlier investigations done by Bacon and Farmer, (1968), reported the presence
of a predominantly β-(1,6)-glucan and several types of β-glucans in the yeast cell wall
before a detailed fractionation of the cell wall components and their characterization
carried by Fleet and Manners, (1976) confirmed the major component of yeast β-glucan
to be a backbone chain of high-molecular β-(1,3)-D-glucan and the minor component to
be β-(1,6)-linked glucose residues.
3.5 Properties of β-glucans
Glucans are characterized mainly by homopolymers of glucose, the chain length, the kind
of glycosidic linkage and the degree of branching. They consist of linear (1,3)-β-linked
backbones with either (1,6) or (1,4)-linked side chains of varying length and
interval/distribution, which form complex tertiary structures stabilized by interchain
hydrogen bonds. The primary structure, solubility, degree of branching (DB), molecular
weight (MW), and helical conformation (triple helix, single helix or random coil
conformation) are characterizing parameters of β-(1,3)-D-glucan that have been shown to
play a role in glucans biological activity (Bohn and BeMiller, 1995; Sandula et al., 1999;
Borchers, et al. 1999).
The solubility of the glucan in water depends on the frequency and length of the side
chains (Laroche and Michaud, 2007). For example, Yeast glucan from Saccharomyces
cerevisiae is insoluble in alkali and water as it is made up of single β-D-glucopyranosyl
units on every eight main chain units (Bacon et al., 1969), which cause the glucan to be
insoluble in alkali and hence named particulate (“insoluble”) β-glucans.
The frequency and side chain units has also been observed to influence the solubility of
schizophyllan, (from Schizophyllum commune fungus) which has a 1,6-D-glucose side
chain on every third glucose unit along the main chain (Figure 3.6a), making it
completely water soluble (Bae et at. 2004, Okobira et al., 2008). On the other hand,
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 40
Figure 3.6: (a) Repeating unit of schizophyllan. (b) Representative model of the triple
helix with the balls representing the side chains (Bae et al., 2004).
Curdlan, a linear β-(1,3)-D-glucan from bacteria, Alcaligenes faecalis, which has no side
chains, forms water-insoluble triple helical structure in aqueous solution. Depending on
the heating temperature, time of heat-treatment and concentration, curdlan has the ability
to form gels with various strength and thus used as thickening and gelling agent in foods
(Harada et al., 1968; Williams, 1991; McIntosh et al., 2005; Laroche and Michaud.,
2007).
In order to improve solubility of particulate (“insoluble”) β-glucans, chemically modified
particulate β-glucans, i.e. carboxymethylated, sulfonated and phosphorylated β-glucans
have also been developed. This has led to the formulation of derivatized particulate β-
glucans that can be administered parenterally compared to particulate (“insoluble”) β-
glucans which causes adverse effects like inflammation, granuloma formation, emboli
formation and pain when administered by parenteral routes (Sandula et al., 1999;
Zekovi´c et al., 2005; Kanlayavattanakul.M. and Lourith.N, 2008).
The differences between β-glucan linkages and chemical structure are also important as
far as solubility, mode of action, and overall biological activity are concerned (Bohn and
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 41
BeMiller, 1995; Toshiro and Zasshi, 2000; Novak and Vetvicka, 2008). The branches
derived from the glycosidic chain core are highly variable and the two main groups of
branching are -(1,4) or (1,6)- glycosidic chains. The alignments of branching follow a
particular ratio and branches can arise from branches (branch-on-branch).
The branching assignments also appear to be species specific, for example, β- glucans of
fungus have (1,6)- side branches whereas those of bacteria have (1,4)- side branches
(McIntosh et al., 2005).
The helical conformation of β-(1,3)-glucans, which includes triple helix, single helix, and
random coil conformers, is recognized as an important contributing factor in biological
activity. β-(1,3)-glucan chains conform to a shape comparable to a flexible wire spring
(Figure 3.6b), that can exist in various states of extension (Klis et al., 2002). Various
studies conducted have confirmed that β-glucans assume conformational change into
triple helix, single helix or random coils in aqueous solution (Takeda et.al. 1978;
Williams, 1991; Bohn and BeMiller, 1995; Sandula et al., 1999; Bae et at. 2004; Zhang et
al., 2005; Surenjav et al., 2006; Okobira et al., 2008). In addition Thammakiti et al., 2004
found that, β-glucans have high apparent viscosity, water holding, oil binding, and
emulsion stabilizing capacities. The triple helix conformation (figure 2.6b) is stabilized
by hydrogen bonding at the C-2 hydroxyl, (Deslandes and Marchessault, 1980; Tadashi et
al., 2008). Researchers have demonstrated that, the immune functions of β-glucans
depend on their stable helix conformational complexity (Zhang et al., 2007).
3.6 Immune-enhancing properties of glucans
The role of Yeast-derived β-(1,3)-glucans as biologically active immunomodulator has
been widely documented. Interest in the immunomodulatory properties of these
polysaccharides was initially raised after investigations done by Pillemer and Ecker,
(1941) using baker’s yeast, Saccharomyces cerevisiae to demonstrate that, a crude yeast
cell wall extract (Zymosan) was responsible for stimulation of macrophages via
activation of the complement system.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 42
Yeast-derived β-(1,3)-glucan is classified as ‘Biological Response Modifier’ (BRM),
which means, it modifies the host’s biological response by stimulating the immune
system (Bohn and BeMiller, 1995; Sandula et al., 1995; Rice et al., 2002; Underhill,
D.M., 2003). The β-glucans stimulate the host’s defense mechanisms against disease
challenge rather than attacking the infective or tumor-causing agent, and therefore
remains nontoxic to the cells of the host organism. Biological response modifiers can be
grouped into two categories according to their effects: cytokines, which are responsible
for communication between immune system cells and regulation of the system, and
immunomodulators, which are responsible for immunopotentiation (i.e. positive
maneuver) or immunosuppression (i.e. negative maneuver) of the immune system (Novak
and Vetvicka, 2008).
The exact mechanism of action of glucans remain to date undecided in spite of long
history of research as biological modulators and search for optimal chemical
configuration is still going on (Freimund et al., 2003; Brown and Gordon, 2003; Zhang et
al., 2007; Vetvicka et al., 2008; Novak and Vetvicka. 2009). As β-glucans are natural
products in origin, they present a complex mixture of ingredients, each one of which may
contribute to their biological activity and thus render challenges in characterization
(Novak and Vetvicka, 2008). However, many researchers concur with the conclusion
drawn on the structural requirements for a physiological effect of β-(1,3)-D-glucans that,
the immunopotentiating activity depends on specific molecular structure such as primary
structure, molecular weight, branching patterns, solubility and helix conformational
features. Immunopotentiation effected by binding of β-(1,3)-glucan molecule or particle
includes activation of cytotoxic macrophages, helper T cells, and Natural Killer (NK)
cells, promotion of T cell differentiation, and activation of the alternative complement
pathway (Bohn and BeMiller, 1995; Cleary et al., 1999; Ooi VE, and Liu, 2000;
Freimund et al., 2003; Zhang et al., 2005).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 43
3.6.1 Interaction of β-glucans with the gastro-intestinal tract
Absorption of orally taken yeast-derived β-(1,3)-glucans and similar compounds has been
reported to be facilitated by enterocytes across the intestinal cell wall into the lymph
where they interact with macrophages to activate immune function (Frey et al., 1999). M
(Microfold) cells within the Peyer’s Patches (Figure 3.7) transport the insoluble whole
glucan particles into the Gut-Associated-Lymphoid Tissue (GALT) (Hong et al., 2004).
Figure: 3.7: section of intestinal lumen showing M (Microfold) cells with the Peyer’s
Patches (Volman et al., 2008)
3.6.2 Immunopharmacological activities of β-glucans
Numerous reports have documented the ability of β-(1-3)-glucans to nonspecifically
activate cellular and humoral components of the host immune system. Yeast-derived β-
(1,3)-glucans and β-glucans from mushrooms work in part by stimulating innate anti-
fungal immune mechanisms to increase host’s resistance to fight a range of pathogenic
challenges from bacteria, fungi, parasites, viruses and cancer (Lindequist et al. 2005).
Radioprotective and adjuvant effects of yeast-derived β-(1,3)-glucans have also been
reported. The mammalian innate immune system can recognize β-glucans as pathogen-
associated molecular patterns (PAMPs) since they are not found in mammals. PAMPs
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 44
represent small molecular sequences of microbial products, usually found in the cell wall
of microorganisms like carbohydrates, lipids, and proteins that are not produced by the
host and are recognized by professional Antigen Presenting Cells (APC) like dendritic
cells (DC) and macrophages by Toll-like receptors (TLR) and other pattern recognition
receptors (PPR) (Kougias et al. 2001; Rice et al., 2002; Brown and Gordon, 2003).
3.6.2.1. Antitumor activity
The ability of glucans to inhibit tumour growth in a variety of experimental tumour
models is well established. A wide variety of mushroom species have been investigated
for their antitumor activities since Japanese researchers began to investigate
systematically whether mushrooms, both wild and cultured, contained substances able to
inhibit tumor growth in the late 1960s. Studies in the host animal suggest that the tumor
inhibiting activity is contained in the polysaccharide fraction and the activities are related
with molecular weight, degree of branching and higher (tertiary) structure of β-glucans.
The triple helical conformation plays an important role in enhancement of the antitumor
activities (Bohn and BeMiller, 1995; Borchers et al., 1999; Sandula et al., 1999; Zekovi´c
et al., 2005; Surenjav et al., 2006).
Some of the isolated compounds with most effective anti-tumor activity include Lentinan
from Lentinus edodes, Schizophyllan from Schizophyllum commune, Grifolan from
Grifola frondosa, and SSG from Sclerotinia sclerotiorum (Suzuki et al. 1987; Ohno et al.
1987; Ooi VE, and Liu F, 2000; Zhang et al., 2005; Zhang et al., 2007). Yeast β-(1,3)-
glucan derived from Saccharomyces cerevisiae (Baker’s yeast) and administered orally in
mice has also demonstrated to inhibit the growth of cancer cells in vivo (Vetvicka et al.,
2002; Hong et al., 2004).
3.6.2.2 Macrophage–activating ability
Several in vitro and in-vivo studies have shown that β-glucans act on several immune
receptors including Dectin-1, complement receptor (CR3) and TLR-2/6 and trigger a
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 45
group of immune cells including macrophages, neutrophils, monocytes, natural killer
(NK) cells, and dendritic cells (Rice et al., 2002; Brown et al., 2003; Brown and Gordon,
2003; Taylor et al., 2002; Taylor et al., 2007; Novak and Vetvicka, 2008; Chan et al.,
2009). They also activate T cells and their cytokine production and hence, both innate
and adaptive response are modulated by β-glucans and phagocytosis enhanced (Vetvicka
et al., 1996; Borchers et al., 2008; Volman et al., 2008). Following oral administration in
animal studies, β-glucans enter the proximal small intestine and some are captured by the
macrophages and then internalized and fragmented into smaller sized β-glucan fragments
within the macrophages. They are transported by the macrophages to the marrow and
endothelial reticular system. The small β-glucans fragments are eventually released by
the macrophages and taken up by the circulating granulocytes, monocytes or
macrophages via the complement receptor (CR)-3 leading to various immune response
(Underhill, 2003; Hong et al., 2004) (Figure 3.8).
Figure 3.8: The uptake and subsequent actions of β-glucan on immune cells (Chan et al,
2009).
3.6.2.3 Radioprotective activity.
The radioprotective (bone marrow protective effect) activities of yeast-derived β-glucans
has been well-established and reported in literature and its uptake is via M cells in the
intestinal Peyer’s patches (Figure 3.7). The oral uptake of yeast-derived β-glucan
particles lead to β-glucan presentation to macrophages in the underlying GALT which is
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 46
then transported into the organs of the reticuloendothelial system –RES (lymph nodes,
spleen and bone marrow) and thus enhancing resistance to microbial invasion,
accelerating bone marrow recovery, restoration of cell formation (hemopoietic recovery)
and increased survival after exposure to radiations following injury as demonstrated in
irradiated mice studies due to prolonged macrophage function (Patchen and MacVittie,
1983; Patchen and MacVittie, 1986; Patchen et al. 1987; Patchen et al. 1998; Wakshull
et al. 1999; Allendorf et al., 2003; Hong et al., 2004; Gu et al. 2005; Cramer et al. 2006;
El-Batal et al., 2008).
3.6.2.4 Cholesterol & glycaemia lowering effects
Research also reports that yeast-derived β-glucan fiber are helpful in lowering elevated
levels of Low Density Lipoprotein (LDL) cholesterol concentrations and in reducing
elevated blood sugar levels. The orally taken beta-glucan has a natural nutritional fiber
property and passes the stomach unchanged. They are not degraded due to lack of
specific human enzyme (β-1,3-glucanase) that would break it to glucose or di-glucose so
that can be absorbed through the intestinal wall. The presence of an unabsorbed high-
fiber diet in the intestine increase intestinal viscosity and decrease cholesterol absorption,
thereby, promoting cholesterol catabolism and excretion of bile acids and thus imparts a
hypocholesterolemic effect. β-glucan as a soluble fiber, delays the digestion of dietary
carbohydrates that can have a possible sugar control benefit for a diabetic patient due to
this delay (Felix et al., 1995; Brennan et al., 1996; Nicolosi et al., 1999; Frank et al.,
2004).
3.6.2.5 Protection against infections
In addition to enhancing the activity of the immune system, β-(1,3)-glucan is also
reported to help prevent infections and aid in wound healing. The anti-inflammatory
activity of β-(1-3)-glucan has been tested in both animal models and in human for
bacterial, fungal, (Meira et al., 1996) protozoal (Tuwaijri et al. 1987) and viral (Ara et al.,
2001) infection. β-(1-3)-glucans have been evaluated in experimental models of
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 47
Escherichia coli, Staphylococcus aureus, Streptococcus pneumonia, ( Hetland et al.,
2000); Pseudomonas aeruginosa, (Koike, 1976); Mycobacterium leprae (Rayyan and
Delville,1983) and Mycobacterium bovis (Hetland et al, 1998) infection. In each case,
treatment with β-(1,3)-glucan had a beneficial protective effect in reducing mortality or
decreasing bacterial counts in infected or susceptible animals.
3.6.2.6. β-(1,3)-glucan as an Adjuvant for vaccine
Oral and intradermal administered yeast β-(1,3)-glucan has been demonstrated to serve as
a vaccine adjuvant with both conducted experiments suggesting that protein antigens can
be conjugated to yeast β-(1,3)-glucan to provide an adjuvant effect for stimulating the
antibody response to protein antigens (Berner et al., 2008).
Other studies which have provided evidence for the beneficial adjuvant effects of yeast β-
(1,3)-glucan are explained below. Vetvicka et al., (2002) demonstrated that, orally-
administered yeast β-(1,3)-glucan had significant effects as a prophylactic treatment to
reduce the mortality of anthrax infection in mice when infected subcutaneously with a
lethal dose of anthrax spores. In addition, the same type of treatment also inhibited the
growth of cancer cells in vivo. The mechanism of action involved the stimulation of three
important cytokines: Interleukin-2, (IL-2), Interferon- γ (gamma), (IFN- γ), and Tumour
Necrosis Factor- α (alpha), (TNF- α). Ara et al., (2001) conducted a study on the adjuvant
effect of zymosan on human immunodeficiency virus type-1 (HIV-1)-specific DNA
vaccine by intramuscular coinjection to BALB/c mice with candidate DNA vaccine for
HIV-1 and suggested to be an effective immunological adjuvant in DNA vaccination
against HIV-1. They suggested that zymosan-mediated DNA vaccination enhanced
helper T cell (Th) 1-mediated immunity and the effect was based on its recruitment and
activation of macrophages, dendritic cells or antigen-presenting cells (APC) through
complement activation. After being activated by zymosan, macrophages induced
production of cytokines such as interferon- γ (IFN-γ), interleukin (IL)-2, IL-8, and
tumour necrosis factor- α (TNF-α).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 48
The ability of β-glucan to exhibit significant adjuvant activity has also been studied and
demonstrated by Wiliams et.al (1989) when they investigated the activity in
immunization against Trypanosoma cruzi, (causative agent of Chagas' disease, that
infects humans and animals in tropics). Mice subcutaneously immunized with particulate
glucan combined with a vaccine of glutaraldehyde-killed T. cruzi reported to show an
increased survival versus controls following days of post-challenge.
β-glucan has also been shown to exert a strong adjuvant effect in experimental
immunization when injected simultaneously with Formalin-killed erythrocytic stages of
the rodent malaria parasite Plasmodium berghei. Antigen-glucan immunization
demonstrated degree of immune protection to the host against the challenge with live
parasite (Holbrook et al, 1981; Maheshwari and Siddiqui, 1989). Also an adjuvant effect
in experimental immunization trials has been observed in visceral/cutaneous
leishmaniasis, disease caused by protozoan parasites Leishmania that infect mammalian
macrophages, and cause visceral infection of the reticulo-endothelial system (Tuwaijri et
al. 1987). Oral glucan administration has also been demonstrated to increase survival in
mice challenged with Staphylococcus aureus or Candida albicans (Rice et al., 2005). In
summary, there is research evidence to substantiate β-glucan’s potential as an adjuvant
for vaccine delivery.
3.7 Oral delivery of vaccines and delivery systems
Prevention of infections by vaccination has proven to be an efficient and cost-effective
strategy for public-health improvement (WHO/IVB/2009). The majority of the available
vaccines are administered by intramuscular or subcutaneous injection, which makes mass
immunization costly and less safe, particularly in developing countries as it poses safety
risks like needle-stick injuries and the transmission of blood-borne diseases. Hence
administration of vaccines via oral route will overcome these problems and have large
implications since the access to trained medical staff to administer vaccines by injection
is limited. Administration of vaccine by oral route could also stimulate immune response
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 49
at mucosal surfaces which is not observed in parenteral vaccination (Lavelle, 2005;
Simerska et al., 2009).
Induction of protective immunity at the mucosal sites will prevent colonization and
translocation of the pathogens across the mucosal barrier since most infectious
microorganisms get entry into the systemic circulation through mucosal surfaces.
Therefore, oral delivery of vaccines for mucosal immunisation would provide a first line
of defence and the potential to prevent systemic infections and at the same time
generating secretory IgA (sIgA), which play a major role in mucosal defense responses at
all mucosal sites apart from the administration site. Hence oral vaccine has the potential
to induce both systemic and mucosal immunity (Challacombe et al. 1992; Shalaby,
1995).
Other advantages that oral vaccines offers compared to parenteral ones are as follows.
Oral administration of vaccines is painless, and therefore pains associated with injections
like site reactions are avoided. It is less invasive and less expensive than injectable ones
and easy to administer outside formal clinical settings without the need for skilled
personnel. It involves only swallowing and hence no possible transmission of infections
from contaminated needles and syringes contrary to injectable ones where the risk of
infectious disease transmission is possible like blood-borne viral diseases due to use of
contaminated needles, reuse and disposal. The ease of oral administration improves
vaccine coverage in remote areas and enhances compliance, particularly in children and
when multiple doses of vaccines are required (Chen and Langer, 1998). Oral vaccines
may be supplemented with adjuvants (Jain et al., 1996) and also may be cheaper to
produce as a result of less tough regulatory requirements for preparations compared to
injectable ones (Mutwiri et al., 2005). Therefore, oral vaccines are seen as effective
prophylactic tool because of better compliance and ability to generate both mucosal and
systemic immunity, whereas injectable vaccines generate only systemic immune response
(Aziz et al., 2007).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 50
Despite all these potential advantages, less focus has been paid on the development of
oral vaccines since the route has been regarded historically to be less effective, as vaccine
antigens undergo digestion in the GI tract before inducing an immune response. Another
concern is that, the mucosal tissues of the gut is continuously exposed to large amounts of
normal flora, microbial and food antigens, and in that respect the gut is rather immune
tolerant whereby antigens can be recognized as food antigens or normal flora instead of
eliciting protective immune responses (Mutwiri et al., 2005; Ilan, 2009). Therefore,
vaccines to be administered orally have to overcome several barriers including
significant dilution and dispersion in the GIT; competition with various live replicating
bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before
reaching the target immune cells (Yeh et al., 1998).
At present, there are limited number of oral vaccines licensed for human use including
polio, cholera, typhoid, rotavirus and adenovirus vaccines (Ogra et al., 2001; Aziz et al.,
2007). Most of these available oral vaccines are composed of attenuated live
microorganisms that can replicate in the mucosa and elicit a sustained immune response
(Vajdy et al 2004). Furthermore, residual toxicity by inactivation that might be present
and possible induction of disease via reversion or mutation to an infectious agent to
people with impaired immune response following administration of attenuated live
vaccines, has changed the focus on vaccine development (Ryan et al., 2001; Perrie,
2006).
Therefore, efforts have focused on the development of subunit vaccines (nonreplicating
antigens) that can be delivered orally by various delivery systems. Sub-unit vaccines are
reported to have poor immunogenic response than traditional vaccines although they
offer a much safer alternative (Lavelle, 2005). Traditional vaccines produce strong
immunogenic response because often contain many components, partly reactogenic
contaminants such as bacterial DNA, RNA or LPS in whole cell vaccines that can elicit
additional T cell help or function as adjuvants, whereas in sub-unit vaccines, some of
these components have been eliminated (Gupta and Siber., 1995; Moingeon et al., 2002;
Wack and Rappuoli, 2005; Petrovsky, 2008). However, the use of adjuvant systems has
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 51
proven to enhance the immunogenicity of these sub-unit vaccines through protection (i.e.
prevent degradation of the antigen in vivo) and enhanced targeting of the antigens to
professional antigen-presenting cells and therefore calls for more development of
vaccine adjuvants (Brayden, 2001; Perrie et al., 2008; Harandi et al., 2010).
Therefore, in order to develop oral vaccines, two important features would be required:
(1) Appropriate delivery systems that would protect the antigens to be delivered orally
from the harsh environment of the gastrointestinal tract (GIT) (e.g. acid proteolytic
enzymes, bile, temperature, and mucus), as well as enabling antigen uptake from the GIT,
and antigen-presentation to appropriate cells of the immune system for the generation of
desired immune responses
(2) Potential adjuvants which can stimulate the immune system to initiate appropriate
immune responses against the delivered antigens (Zhou and Neutra, 2002; Simerska et
al., 2009).
3.7.1 Roles of adjuvant
When conjugated to vaccines, adjuvants can enhance the immunogenicity of highly
purified or recombinant antigens; can reduce the amount of antigen or the number of
immunizations needed for protective immunity; can improve the efficacy of vaccines in
newborns, the elderly or immune-compromised persons and can be used as antigen
delivery systems for the uptake of antigens by the mucosa (Gupta and Siber., 1995;
Petrovsky and Aguilar, 2004; Aguilar and Rodriguez. 2007; Rajput et al., 2007).
Adjuvants can be broadly separated into two categories based on their principal
mechanisms of action:
(i) Particulate antigen delivery systems, (e.g., emulsions, microparticles,
Immunostimulating complexes (ISCOMs), virus-like particles (VLPs) and liposomes)
and function mainly to target associated antigens into antigen-presenting cells.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 52
(ii) Immunostimulatory adjuvants derived predominantly from pathogens and often
represent pathogen-associated molecular patterns (e.g., lipopolysaccharide,
monophosphoryl lipid A, CpG DNA), which activate cells of the innate immune system
(Gupta and Siber, 1995; Singh and O’Hagan. 2002; Moingeon et al., 2002).
Several authors have reported different classes of adjuvants that have been evaluated for
enhancing immune responses to vaccines which are summarized in Table 3.2 (Gupta and
Siber., 1995; Chen and Langer, 1998; Singh and O’Hagan. 2002; Moingeon et al., 2002;
Zhou and Neutra, 2002; Rajput et al. 2007; Petrovsky, 2008).
Table 3.2: Examples of Different Classes of Adjuvants That Have Been Evaluated for
Enhancing Immune Responses to Vaccines
MINERAL SALTS Aluminum salts (hydroxide, phosphate, alum)
Calcium phosphate
IMMUNOADJUVANTS
Cytokines e.g. IL-2, IL-12,
Granulocyte-macrophage colony stimulating factor (GM-CSF)
EMULSIONS
MF59 (stabilized squalane/sater)
QS21 (purified saponin from barks of Quillaja Saponaria tree)
Incomplete Freund adjuvant (IFA), without the killed mycobacteria)
NATURAL/SYNTHETIC BACTERIAL PRODUCTS
Monophosphoryl lipid A (MPL)
RC-529 (synthetic MPL-like acylated monosaccharide)
Holotoxins (cholera toxin (CT); Lymphotoxin (LT) from Escherichia coli, Pertussis toxin (PT)
Bacterial DNA, cytidine-phosphate-guanosine (CpG) oligonucleotides`
MICROPARTICLES/ PARTICULATE ADJUVANTS
Liposomes
Virosomes
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 53
Chitosan
ISCOMs
PLGA (Poly-(D,L)-lactide-co-glycolic acid)
Microparticles offer various significant advantages as drug delivery systems, including:
(i) An effective protection of the encapsulated active agent against (e.g. enzymatic)
degradation.
(ii) The possibility to control the release rate of the incorporated drug over periods of
time and (iii) an easy administration (O'hagan, 1996; .Siepmann and Siepmann, 2006).
3.7.2 Oral vaccine delivery systems
Delivery of a vaccine via the oral route in a sufficient dose to induce a protective immune
response depends on overcoming the loss of antigen integrity that occurs during intestinal
passage. Several strategies have been employed to prevent this loss of antigenicity and
improve the delivery of vaccines by the oral route. These include live vectors, plant-
based edible vaccines system, and particulate formulations such as microparticles,
liposomes, and virus-like particles (Litwin et al., 1997; Ellis, 2001; Moingeon et al.,
2002; Lavelle, 2005). Enteric coated vaccine systems have also been explored as another
strategy for delivering oral vaccines by protecting the vaccine from harsh environment of
the GIT and release the delivered vaccine in unencapsulated form in the intestine
following dissolution of the enteric polymer film (Jain et al., 1996; Snoeck et al., 2003;
Huyghebaert et al., 2005).
In order for an effective immune response to be mounted against orally administered
antigens, the antigen must be taken up and presented to the immune system through M
cells of the Peyer’s patches present in the small intestine (Figure 3.7) that function to
sample the intestinal contents and present them to underlying gut-associated lymphoid
tissue (GALT) (Chen and Langer, 1998; Clark et al., 2001; Lavelle, 2005; SSilin et al.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 54
2007). The GALT consist of inductive sites (where antigens are encountered and
responses are initiated) and effector sites (where local immune responses occur) and is
linked by a homing system, whereby cells induced by antigen in the GALT migrate to the
circulation and, subsequently, colonize the mucosa and thus induction of immune
response locally in the gut and at distant mucosal sites, as well as systemic humoral and
cellular immune responses by oral vaccination (Yeh et al., 1998; Clark et al., 2001;
Lavelle, 2005).
For the successful delivery of antigens by the targeted oral vaccine carrier, a number of
considerations have to be taken into account as reported by many authors. To mention
few but not exhaustive, these include protection of antigens from intestinal metabolism,
high entrapment efficiency in particulate formulation, stability of antigen retained in the
particle, uptake of vaccine by M cells through targeting and/or adjuvants and particle size
(Table 3.3) of carrier system (O'hagan, 1996; Thomas et al., 1996; Yeh et al., 1998;
Brayden et al., 2005).
Table 3.3: Sites, mechanisms and particle sizes proposed to be involved in the uptake of
particles.
Site mechanism Particle size
Villus tips persorption 5-150 µm
Intestinal macrophages phagocytosis 1µm
Enterocytes endocytosis < 200 nm
Peyer’s patches transparacellular < 10 µm
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 55
3.8 Materials and Methods
Presented in this chapter are the laboratory methods carried out for the extraction and
isolation of yeast glucan from baker’s yeast cells (Saccharomyces cerevisiae), followed
by encapsulation to form cationic yeast carriers, antigen loading into yeast carriers,
formulation of pellets and minitablets containing antigen loaded in yeast carriers/shells
(yeast-glucan) and enteric coating of pellets and characterization of these dosage forms.
Materials and equipment used for the extraction and purification of yeast-glucan from
baker’s yeast cells (BRUGGEMAN instant) are listed in Table 3.4a and 3.4b.
Table 3.4a: Materials used for the extraction and purification of baker’s yeast cells
Name of material Supplier
Baker’s yeast cells, (Saccharomyces cerevisiae), BRUGGEMAN instant
Algist Bruggeman N.V. Gent –Belgium
Sodium hydroxide pellets VWR® International, Leuven.
Hydrochloric acid 37% VWR® International, Leuven.
Distilled water Pharm-tech lab. Ghent univ.
Disinfectol® solution (Ethanol denaturated with up to 5% Ether) Acetone
Chem-Lab, Belgium LAR BLOEI, Belgium
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 56
Table 3.4b: Equipment used for the extraction and purification of baker’s yeast cells
Equipment Supplier
Glass bottle, 2000ml SCHOTT, Germany
Balance Mettler-Toledo, Switzerland
Heater stirrer Heidolph, Germany
Magnetic stirrer LABINCO, Belgium
Thermometer, 0-200°C
pH meter Consort, Belgium
Centrifuge Heraeus, Germany
3.8.1 Extraction and purification of yeast-glucan from Baker’s yeast,
(Saccharomyces cerevisiae).
Extraction and purification of yeast-glucan from baker’s yeast cell walls (Saccharomyces
cerevisiae) was carried out according to the reported procedure for isolating fungal-
derived β-D- glucans involving treatment with hot alkali and acids (Bacon et al. 1969;
Sandula et al., 1999; Soto and Ostroff, 2008).
One liter solution of one molar Sodium hydroxide (1M NaOH) was prepared by
dissolving 40g of sodium hydroxide pellets and stirred at 80°C. One hundred gram of
Baker’s yeast (Saccharomyces cerevisiae) (BRUGGEMAN instant) was then suspended
into this preheated solution and the reaction mixture was continuously stirred gently and
maintained at a temperature of 80°C for 1 hour (Figure 3.9).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 57
The mixture was centrifuged at 2000 x g for 10 minutes to collect insoluble material
containing yeast cell wall (shells) components. The centrifuged insoluble material was
then suspended in 1 liter of distilled water and its pH was corrected from 12 -13 to 4-5
using conc. HCl.
Figure 3.9: Preheating of 1M Sodium Hydroxide solution and Baker’s yeast cells
(BRUGGEMAN instant) for extraction process.
The reaction mixture was allowed to heat up to 55°- 60°C and incubated/maintained at
this temperature for 1hour (Figure 3.10).
Figure 3.10: Incubation of yeast cell-wall extracts in water at 55°- 60°C with adjusted
pH 4-5.
The obtained suspension was centrifuged at 2000x g for 10 minutes to collect insoluble
cell-wall material (sediment/precipitate) and the supernatant was discarded. The sediment
was washed once with one liter of distilled water, and the precipitate was collected by
centrifugation at 2000x g for 10min. The precipitate was again washed thrice with 800ml
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 58
of Disinfectol® solution (ethanol denaturated with up to 5% ether) and the sediment
collected by centrifugation at 2000x g for 10min.
The sediment was dried in an oven at 40°C for 24hr. After grinding in a mortar and
pestle, the yield was 9.45g of fine slightly off-white powder or 9.45% of the total weight
of the yeast cells used and was designated as yeast-glucan shells or simply yeast shells
(Figure 3.11).
Figure 3.11: Extracted dried yeast cell-wall components (yeast-glucan shells).
Figure 3.12: Schematic overview of the extraction and isolation of yeast-glucan from
Baker’s yeast cell walls.
100g of Baker’s yeast cells (S.
cerevisiae).
Extraction with 1MNaOH, 80°C, 1hr Centrifugation at 2000 x g;
10 min
Sediment, + 1 L distilled water treatment, + Conc HCl to pH= 4-5; stirred at heat 55°-60°C for 1hr, centrifugation at 2000x g; 10 min
Supernatant discarded
Supernatant discarded
Supernatant discarded
Yeast glucan dry powder (yeast shells)
Sediment, + 1 L distilled water treatment, once washing, centrifugation at 2000x g; 10 min
Sediment, + 800ml Disinfectol® treatment; 3 times washing; centrifugation at 2000x g; 10 min
Sediment, oven drying at 40°C/ 24hr
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 59
3.8.2 Antigen loading into yeast carriers
3.8.2.1. Preparation of cationic yeast carriers
Yeast-glucan cationized carriers were developed using layer-by-layer (LBL) assembly of
yeast-glucan shells suspended and held together by electrostatic interactions between
polyelectrolyte solutions of anionic charged tRNA genetic material and cationic charged
Polyethylenimine (PEI) polymer, each separately dissolved in Tris HCL-EDTA-NaCl
(TEN) buffer solution (Soto and Ostroff, 2008). Materials used for the preparation of
Cationic yeast carriers are listed in Table 3.5.
Table 3.5: Materials used for the preparation of Cationic yeast carriers
Name of material Supplier
TRIZMA® HYDROCHLORIDE (Tris [hydroxymethyl] aminomethane hydrochloride),
Sigma Aldrich
Polyethylenimine (PEI) polymer, water-free branched, (25kDa) Sigma Aldrich
Ribonucleic acid from Torula utilis yeast, (tRNA) Sigma Aldrich
Ethylenediamine tetraacetic acid disodium salt hydrate (EDTA) Sigma Aldrich
Sodium Chloride VWR® International, Leuven
In order to form cationic yeast carriers that could allow antigen loading, the following
solutions were preliminarily prepared so that the yeast shells could be suspended in
during layer-by-layer formation.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 60
3.8.2.2. Preparation of Buffer solution, Tris HCL-EDTA-NaCl (TEN) solution
7.88g of Tris HCl (Mw. 157.6), 0.744g of EDTA (Mw. 372); and 8.766g of NaCl (Mw.
58.44) were weighed and dissolved in distilled water to produced 1 liter buffer solution
with 50mM Tris HCl, 2mM EDTA, and 0.15mM NaCl (TEN) concentration. The pH of
TEN buffer solution was adjusted to pH 8.0 using 1M NaOH solution.
3.8.2.3. Preparation of anionic core polymer tRNA solution
1g of tRNA was weighed and dissolved in 100ml of buffer TEN solution to make
10mg/ml anionic core polymer tRNA in TEN.
3.8.2.4. Preparation of cationic PEI (Polyethylenimine) solution
200mg of PEI was weighed and dissolved in 100ml of buffer TEN to make 2mg/ml
cationic PEI in TEN.
3.8.2.5. Preparation of cationic yeast carriers
10g of yeast shells was mixed with 50ml of the anionic core polymer tRNA (10mg/ml in
TEN) to minimally hydrate the powder particles. The resultant slurry was incubated for 2
hours at room temperature to allow the particles to swell and adsorb the tRNA solution.
Sufficient amount of cationic PEI (2mg/ml in TEN), approx. 150 ml, was added to form
yeast-glucan shells encapsulated tRNA polyplexes, and the particles were resuspended by
homogenization/stirring for 1h to allow PEI adsorption and microcomplex formation.
Sufficient distilled water was added to the resultant suspension to obtain approximately
500 ml. This mixture was centrifuged at 2000 x g for 10 minutes and supernatant
discarded.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 61
The washing procedure with distilled water was repeated three times each at a
centrifugation speed of 2000 x g for 10 minutes resulting into the collection of sediment
of yeast-glucan cationized carriers and supernatant discarded.
Figure 3.13: Schematic overview of the preparation of cationic yeast-glucan carriers.
3.8.2.6. Loading of Bovine Serum Antigen to Cationic yeast carriers
Cationic yeast-glucan carriers were loaded with Bovine Serum Albumin (BSA), chosen
as the model protein antigen in this work in the ratio of 1:1; i.e. 1g of BSA was loaded
with 1g of cationized yeast-glucan. The following were the procedures carried out to load
the BSA antigen into cationic yeast-glucan carriers.
3.8.2.6.1. Preparation of Phosphate-buffered Saline (PBS buffer- 7.4 pH)
Phosphate-buffered Saline was prepared by dissolving the constituents in one liter of
distilled water and its pH adjusted to 7.4 with 1M NaOH solution as listed in Table 3.6.
The buffer was used as a diluent for cationic yeast-glucan carriers and BSA.
10g dry yeast-glucan shells + 50ml tRNA soln (10mg/ml in TEN), incubation 2hr, at room temperature
Add approx. 150ml PEI soln (2mg/ml in TEN), stir for 1 hr, at room temperature.
Wash with distilled water, approx 250ml, 3 times with centrifugation at 2000x g, 10 min each turn.
Sediments of cationic yeast-glucan carriers collected.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 62
Table 3.6: Composition of Phosphate-buffered Saline (PBS buffer- 7.4 pH)
Ingredient Concentration (g/L)
Sodium Chloride, (NaCl) 8.0
Potassium Chloride, (KCl) 0.2
Disodium Hydrogen Phosphate, (Na2HPO4) 1.44
Potassium dihydrogen phosphate, (KH2PO4) 0.24
Distilled water to 1.0
pH (adjusted with 1M NaOH) to 7.4
3.8.2.6.2. Loading of BSA to cationic yeast carriers in PBS solution
Ten gram of BSA antigen was dissolved in one liter of PBS (pH 7.4) and stirred for five
minutes to form a mixture of BSA/PBS buffer solution. This was followed by the
addition of ten gram of cationic yeast carriers to this buffer solution containing BSA,
then, stirred for thirty minutes and incubated overnight in a refrigerator. The mixture was
centrifuged at 2000 x g for ten minutes to collect sediment of loaded BSA/ yeast-glucan.
The collected sediment (loaded BSA/ yeast-glucan) was washed thrice with distilled
water (approx. 800ml) to remove traces of PBS solution and the precipitate was again
collected by centrifugation at 2000 x g for ten minutes. The precipitate was finally
washed thrice with acetone (approx. 200ml) and the sediment collected by centrifugation
at 2000 x g for ten minutes. The sediment was dried in an oven at 25°C for 24 hr forming
dry granules which were then grinded using mortar and pestle to get loaded BSA/yeast-
glucan carriers or loaded BSA/yeast carriers.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 63
Centrifugation, 2000 x g; 10 minutes
Sediment oven-drying at 25°C/ 24 hrs
Figure 3.14: Schematic overview of the loading of cationic yeast-glucan carriers with
BSA.
3.8.3 Confocal laser scanning microscopy
Confocal laser scanning microscopy (CLSM) was used to visualize microscopically the
uptake and attachment of fluorescent labeled ovalbumin (OVA) (used as a model antigen)
to yeast shells.
100 mg of yeast shells was diluted with 500µl of tRNA solution (10mg/ml in TEN
buffer) and vortexed well for 1 minute to form a slurry which was incubated for 30
minutes at room temperature. A small sample of the slurry was scooped to serve as a
control sample before addition of cationic PEI solution. The remaining slurry was diluted
with 5 ml of cationic PEI (2mg/ml in TEN buffer) to form a suspension and vortexed
again for 1 minute, followed by an incubation period of 30 minutes at room temperature.
Sediment washing with approx. 800 ml distilled water, 3 times each with centrifugation, at 2000 x g; 10 min.
Sediment washing with approx. 800 ml Acetone , 3 times each with centrifugation, at 2000 x g; 10 min.
Loaded BSA/yeast-glucan carriers
10g Yeast-glucan carriers + 10g BSA in 1L PBS; stirred 30 minutes, incubated overnight in refrigerator
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 64
The resulting suspension was centrifuged at 2000 x g for 10 minutes to collect the
sediment and washed twice with demineralized water. Loading with OVA was performed
by adding 10ml of OVA solution (10mg/ml in PBS) followed by thorough mixing and
then mounted on the CLSM.
3.8.4. Formulation of fast-disintegrating pellets loaded with Bovine serum antigen
(BSA/yeast-glucan pellets)
3.8.4.1. Pellet Production
Pellets loaded with 5% w/w BSA/yeast-glucan were produced via the
extrusion/spheronization technique. Enzyme resistant starch – type III, modified starch
grade (UNI-PURE® EX starch, National Starch and Chemical Co., Bridgewater, NJ,
USA) was chosen as the main spheronizing aid (DukiC-Ott et al., 2008). Other
ingredients incorporated into the formulation were: hydroxypropylmethylcellulose
(HPMC) (Methocel® E15) as a binder, sorbitol to improve wettability in the formulation
and distilled water as granulating liquid.
Loaded BSA/yeast-glucan carriers, UNI-PURE®EX starch, Methocel® E15 and sorbitol
were dry mixed (batch size: 300g) for 15 minutes using planetary mixer (Kenwood Chief,
Hampshire, UK) with a K-shaped mixing arm to obtain a uniform powder blend. 230g of
distilled water was added at once to the powder mixture in the same mixer and blended
for 5 minutes to form a wet granulated mass ready for extrusion. The wet mass was
extruded axially at a speed of 50 rpm using a single screw extruder (Dome extruder, Lab
model DG-L1, Fuji Paudal, Tokyo, Japan) fitted with dome-shaped screen of 1mm
diameter perforations and 1.2mm thickness. The formed extrudates were spheronized for
5 minutes in a spheronizer (Caleva Model 15, Caleva, Sturminster Newton, Dorset, UK)
with a cross-hatched friction plate at a speed of 850 rpm to form pellets followed by
drying in an oven at 40°C for 24hrs.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 65
Table 3.7: Composition of 5%w/w BSA/yeast-glucan Pellets
Name of Ingredient Quantity (g) Trade name & supplier
Loaded BSA/ yeast-glucan carriers
15.0
Modified (resistant) starch 241.965 UNI-PURE®EX starch; National Starch & chemical co., Bridgewater, New jersey, USA
Hydroxypropylmethylcellulose 13.965 Methocel® E15 LV EP Colorcon, Dartford, UK
Sorbitol 29.1 Sorbidex® P 16616; Cerestar, Vilvoorde, Belgium
Demineralised water 230.0
As release of BSA from the pellets can not be measured spectrophotometrically, we also
made riboflavin (5%w/w) loaded pellets. These “tracer pellets” were coated in the same
batch as the BSA loaded pellets, and afterwards dissolution tests were performed with the
riboflavin pellets in order to evaluate coating efficiency.
Table 3.8: Composition of 5%w/w riboflavin sodium phosphate pellets
3.8.4.2. Coating of pellets with Eudragit® 30L D-55, dispersion 30%w/w.
A total weight of 400g pellets (300g riboflavin pellets and 100g of BSA/yeast-glucan
pellets) of size fraction 710 - 1400 µm were mixed together and coated in a fluid bed
Name of Ingredient Quantity (g) Trade name & Supplier
Riboflavin sodium phosphate
15.0
Sigma-aldrich
Modified (resistant) starch
241.965
UNI-PURE®EX starch; National Starch & chemical co., Bridgewater, New jersey, USA
Hydroxypropylmethylcellulose
13.965 Methocel® E15 LV EP Pharm; Colorcon, Dartford, UK
Sorbitol 29.1 Colorcon, Dartford, UK
Demineralised water 230.0
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 66
coating equipment (GPCG1, Glatt, Binzen, Germany) using a bottom spray mode with
Würster insert.
Table 3.9: Composition of the coating dispersion
Name of the ingredient Quantity (g) Function Supplier
Eudragit® 30L D-55,
30% w/w dispersion
357.0
Acid-resistant film forming polymer
Rohm, Darmstadt, Germany
Glyceryl monostearate 9.1 Glidant Federa, Braine-l’Alleud, Belgium
Polysorbate 80 (33% aq.sol) 11.2 Wetting agent/surfactant Alpha Pharma, Nazareth, Belgium
Triethyl citrate (TEC) 21.7 Plasticizer Sigma-Aldrich Chemie, Steinheim, Germany
Demineralised water 301.0 Dispersion medium
3.8.4.3 Preparation of coating dispersion (700g)
11.2g of polysorbate 80 (33% aqueous solution), 21.7g of triethylcitrate and 301g of
water were stir heated to 70-80°C (above the melting point of glycerol monostearate).
Then, 9.1g of glycerol monostearate was added. This mixture was removed from the
heating source and homogenized for 10 minutes using a rotor-stator mixer (Silverson,
Bucks, UK). Lastly, 357g of Eudragit® 30L D-55 was added and stirred gently with a
magnetic stirrer for 30 minutes to homogenize and stabilize the dispersion before starting
the coating process. The dispersion was constantly mixed gently with a magnetic stirrer
throughout the coating process to maintain its homogeneity.
Pellets were pre-heated to 23-26°C prior to suspension spraying while the inlet air
temperature was set between 28 and 30°C in order to maintain a product temperature of
25 - 26°C. The coating dispersion was maintained for 2hrs at a spray rate of 4.0 g/min,
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 67
through a 0.8 mm nozzle using an atomizing air pressure of 1.5 bars. Pellets were left to
dry at the same product temperature (26°C) for 15 min after coating. At the end of the
process, the total weight gain of dry coated pellets was 477g (equiv. to 19.25% w/w dry
polymer weight gain) from an initial weight of 400g.
3.8.5 Formulation of fast-disintegrating minitablets loaded with Bovine serum
antigen (BSA/yeast-glucan minitablets)
Minitablets of 5mm diameter loaded with 5%w/w BSA/yeast-glucan were prepared by
direct compression. Loaded BSA/yeast-glucan and required excipients (Table 3.10) were
dry mixed (batch size: 100g) for 15 minutes in a Turbula® mixer (model T2A, W.A.
Bachofen, Basel, Switzerland) to obtain a uniform powder mixture. The powder blend
was directly compressed into tablets with a compression force of 150-155kgf (kilogram-
force, measured by digital indicator, AD-4532A connected to the tablet press) using a
single punch eccentric tablet press type EKO, Korsch, Berlin, Germany.
Table 3.10: Composition of 5%w/w BSA/yeast-glucan minitablets
Name of the ingredient Quantity (g) Function Supplier
Loaded BSA/ Yeast-glucan pulv 5.0 Active ingredient
Microcrystalline Cellulose, Avicel® PH 101
45.0 Binder FMC, BioPolymer, Little Island, Ireland
Lactose, Pharmacon® DCL 21 45.0 Filler/diluent Pharmacon
Cross-linked polyvinylpyrrolidone, Polyplasdone® XL-10
5.0 Super disintegrant ISP, Switzerland
Magnesium Stearate 0.5 Lubricant Fagron, Belgium
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 68
3.8.6 Evaluation and characterization of pellets and minitablets
3.8.6.1 Pellet size analysis
Sieve analysis method was used to evaluate pellet size and yield, whereby 250g pellets
were sieved for 10 minutes at an amplitude of 3mm on a shaker (Type VE 1000, Retsch,
Haan, Germany) using 1400, 1120, 1000, 800, 710, 500 and 250µm sieves (Retch, Haan,
Germany) in order to remove fines and agglomerates. The pellet yield was calculated
based on the pellet fraction between 710 - 1400µm separated by sieving and presented as
a percentage of the total pellet weight. This size fraction was used for all other
measurements.
3.8.6.2 Pellet shape and size analysis
An image analysis system was used to determine shape and size of coated pellets.
Photomicrographs of pellets were taken with a digital camera (Camedia® C-3030 Zoom,
Olympus, Tokyo, Japan), linked with a stereomicroscope system (SZX9 DF PL 1.5x,
Olympus, Tokyo, Japan). A cold light source (Highlight 2100, Olympus, Germany) and a
ring light guide (LG-R66, Olympus, Germany) were used to obtain top light illumination
of the pellets against a dark surface. The images were analyzed by image analysis
software (AnalySIS®, Soft Imaging System, Münster, Germany). The magnification was
set in a way that one pixel corresponded to 5.7µm and around 100 pellets were analyzed
from the prepared batch. Each individual pellet was characterized by the mean Feret
diameter (FD) (average of 180 calliper measurements with an angle of rotation of 1°),
aspect ratio (AR) (ratio of longest Feret diameter and its longest perpendicular diameter)
and two-dimensional shape factor (eR), as described by Podczeck and Newton (1994).
2
12
−−
⋅⋅=
l
br
Pe
m
R
π (equation. 3.1)
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 69
Where r is the radius, Pm is the perimeter, l is the length (longest Feret diameter) and b is
the breadth (longest diameter perpendicular to the longest Feret diameter) of a pellet. An
average value for all pellets was calculated as the mean pellet size (mean FD).
3.8.6.3 Pellet friability test
10g of pellets (FS, 10g) was placed in an abrasion wheel together with 200 glass beads
(diameter: 4mm) and fitted to a friabilator; (type PTF, Pharma Test, Hainburg, Germany).
The sample was subjected to falling shocks for 10 minutes at a rotational speed of 25 rpm
and fines collected by sieving through 250µm mesh for 5 minutes at 2mm amplitude. The
weight difference was obtained by weighing the pellets retained above 250µm (Fa) and
compared to the initial weight of the sample and percentage of friability determined using
the following equation:
( )( )
100% ∗
−=
Fs
FaFsFriability (equation. 3.2)
3.8.6.4 Pellet disintegration test
The disintegration time for uncoated and coated loaded yeast-glucan pellets was
measured in a disintegrator (Type PTZ, Pharma Test, Hainburg, Germany) using a
method modified from the Eur. Ph. 6th edition monograph for tablet disintegration. Using
a 500 µm mesh placed at the bottom of each tube (6 tubes) and discs to increase the
mechanical stress on the pellets, 100mg of uncoated pellets and coated pellets were
dispersed in each tube separately and immersed in a beaker containing 600ml of
demineralized water for uncoated pellets and phosphate buffer (PB pH 6.8) for coated
pellets respectively as disintegration media (both preheated to 37°C). Results represent
the average of six determinations.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 70
3.8.6.5 Dissolution tests
The dissolution tests were performed using the USP apparatus (VK 8000, VanKel, New
Jersey, USA) with paddles at a rotational speed of 100 rpm, in 900 mL dissolution
medium at 37°C. Acidic dissolution medium (0.1N HCl; pH 1.2) was used during the
first 2h, followed by 1h in phosphate buffer (PB; pH 6.8). During dissolution in 0.1N
HCl, samples of 5 mL were withdrawn from the dissolution vessel at 5, 10, 15, 20, 30,
45, 60, 75, 90, 105 and 120 min and in PB (pH 6.8) at 5, 10, 15, 20, 30, 45 and 60 min.
As mentioned before, the dissolution test was performed with the riboflavin loaded
pellets and the sample amount used for analysis (1g) was adjusted to obtain sink
conditions. The concentration of riboflavin was measured spectrophotometrically at 267
nm in 0.1N HCl and pH 6.8 PB, respectively, by means of a double-beam
spectrophotometer (UV-1650PC, Shimadzu, Kyoto, Japan).
According to the requirements from Eur. Ph. 6th edition, an enteric coat was successfully
applied if less than 10 % of drug is released after 2 h of dissolution in acid dissolution
medium (0.1N HCl).
3.8.6.6 Scanning electron microscopy
The morphology of the coating surface and the coating thickness of pellets were
visualized by scanning electron microscopy (SEM) (Jeol JSM 5600 LV, Jeol, Tokyo,
Japan). Pellets were radially sheared to obtain cross-sections and coated with platinum
using a sputter coater (Auto Fine Coater, JFC-1300, Jeol, Tokyo, Japan) to assure
conductivity. Photomicrographs were taken with a scanning electron microscope and the
coating thickness of a pellet was measured at four sites per pellet.
3.8.6.7 Tablet hardness test
The resistance to crushing of the tablets (n=20) was determined as described in European
Pharmacopoeia, 6.0th edition, using an automated hardness tester, SOTAX HT 10 V1.31,
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 71
equipped with auto alignment device and software (Q-Doc hardness tester software) for
statistical processing. The test jaws constant speed was 1.00 mm/sec. Results were
expressed as the mean, minimum and maximum values of the forces measured, all
expressed in Newtons representing twenty determinations.
3.8.6.8 Tablet friability test
The friability test for BSA/yeast-glucan minitablets was determined according to Eur. Ph.
6th edition, monograph for friability of uncoated tablets.
A sample of twenty tablets (Ws) was weighed and placed on a sieve 1000 µm size to
remove any loose dust with the aid of air pressure before being placed in a friability drum
and fitted to a friabilator (type PTF, Pharma Test, Hainburg, Germany). The sample was
subjected to roll and fall by rotating the drum 100 times and then followed by removal of
any loose dust generated from the tablets during the test. The tablets were weighed again
to determine their final weight (Wf). The friability was expressed as the loss of mass and
was calculated as a percentage of the initial mass using the following equation:
( )( )
100% ∗
−=
Ws
WfWsFriability (equation. 3.3)
3.8.6.9 Tablet disintegration test
The disintegration time for the minitablets was measured in a disintegrator (Type PTZ,
Pharma Test, Hainburg, Germany) using a method from the Eur. Ph. 6th edition
monograph for tablet disintegration. One tablet was placed in each of the six tubes and
discs were added to each tube in order to increase the mechanical stress on the tablets.
The assembly was suspended in a beaker containing 600ml of demineralized water as
disintegration medium preheated to 37°C. Results represent the average of six
determinations.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 72
3.9 Results and Discussion
3.9.1 Laser confocal microscopy
Microscopic observation using confocal scanning laser microscopy showed a successful
binding or entrapment of green-labeled fluorescent ovalbumin (OVA) antigens to yeast
shells upon mixing anionic tRNA and cationic PEI solution (Figure 3.15) as a result of
electrostatic interactions (Li et al., 2008; Soto and Ostroff, 2008).
The Confocal microscopy illustrated how the modification of the yeast shells with tRNA
and cationic PEI allowed a successful loading of the shells with green fluorescent
Ovalbumin (OVA).
Figure: 3.15: Confocal microscopy images showing the interaction between green
fluorescent ovalbumin and yeast shells before and after cationic modification. The bar
corresponds to 20µm. The left column is the green fluorescence image; the middle
column is the transmission image and the right column is the overlay of both green
fluorescence and transmission image.
The first row of Figure 3.15 shows no accumulation of green fluorescence inside the
yeast shells as large excess of green fluorescence is observed in the solution outside the
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 73
yeast shells. Upon modification of yeast shells with tRNA, an identical situation is
observed as shown in the middle row of Figure 3.15.
Contrary, after modification with tRNA and cationic PEI, the green fluorescent OVA
strongly accumulated within the yeast shells leaving the external solution dark as shown
in the lower row of Figure 3.15.
3.9.2 Sieve analysis
The size and weight distribution of sieved pellets (250g) are presented in Table 3.11,
indicating that a pellet yield (710-1400 µm fraction) of higher than 90% could be
obtained. This shows that the addition of a binder (hydroxypropylmethylcellulose) to
UNI-PURE® EX starch was necessary to obtain an acceptable yield since the binder
increased the mechanical strength of wet extrudates and consequently fewer fines were
formed during spheronization (Dukic-Ott, et al., 2007a).
Table 3.11: Results of size and weight distribution of pellets by sieve analysis
Sieve size fraction Weight of sieved pellets (g) Percentage yield (%)
> 2000µm 1.000 0.4
1400-2000 µm 10.605 4.24
1120-1400 µm 64.788 25.92
1000-1120 µm 75.059 30.02
800-1000 µm 86.881 34.75
710-800 µm 7.481 2.99
500-710 µm 3.781 1.51
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 74
250-500 µm 0.397 0.16
< 250 µm - -
3.9.3 Pellet shape and size analysis
The shape of the pellets is characterized by the aspect ratio which describes the deviation
in shape from a circle to an ellipse and by the roundness. The latter is an indication of
surface irregularities. The image analysis results indicated that coated pellets (710-
1400µm) had a Feret mean diameter of 0.967±0.15mm, sphericity (aspect ratio, AR) of
1.2±0.25 and a two-dimensional shape factor (eR), 0.87±0.11 (n=100), falling within the
limits set by Chopra et al. (2002) for optimum pellets shape and flow characteristics.
3.9.4 Pellet friability test
Starch-based pellets had a high mechanical strength, since friability values of less than
0.01% were obtained. A low friability indicates the pellets’ ability to withstand the shear
forces during fluid bed coating.
3.9.5 Pellet disintegration test
The time taken for complete disintegration of uncoated pellets was 5-10 minutes and 10-
15 minutes for the coated pellets in water and PB pH 6.8 respectively. This showed that
modified starch is a good choice as the main excipient for the formulation of pellets with
fast-disintegrating properties and consequently fast drug release in dissolution medium
(DukiC-Ott et al., 2008).
Furthermore, photomicrographs of coated pellets taken with a digital camera (Camedia®
C-3030 Zoom, Olympus, Tokyo, Japan), linked with a stereomicroscope system (SZX9
DF PL 1.5x, Olympus, Tokyo, Japan) showed that the Eudragit® 30LD-55 coated pellets
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 75
did not dissolve when placed in acidic medium but showed dissolution in alkaline
medium (Figure 3.16). These results suggest that Eudragit® 30LD-55 is a good stabilizer
as gastric acid-resistant polymer.
Figure 3.16: Photomicrographs visualizing uncoated and coated BSA/yeast glucan
pellets in acidic and alkaline medium. Time scale: Left column, t = 0 min, the start of
the experiment i.e. when the acid and alkali were added to the pellets. Middle column, t
= 5 min, disintegration and appearance of pellets after five minutes exposure. Right
column, t=10 min, disintegration and appearance of pellets after ten minutes exposure.
3.9.6 Pellet dissolution test
In-vitro drug release profile (Figure 3.17) showed that all riboflavin was released in 30
minutes in phosphate buffer (pH 6.8). Drug release was fast due to disintegration of
UNI-PURE® EX starch as the main excipient which ensures fast exposure of the drug to
the dissolution medium and also due to the inclusion of sorbitol in the formulation which
has high solubility in water (Dukic-Ott, et al., 2007b).
In addition to that, the drug release profile showed that enteric coating was successfully
applied to the riboflavin pellets since after 2h of dissolution in acid dissolution medium
(0.1N HCl), less than 10 % drug was released which is according to the requirements in
European Pharmacopoeia, 6th edition (maximally 10%). The release in 0.1N HCl after 2h
(4.4 %) was below the limits indicated in the European Pharmacopoeia.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 76
Mean dissolution profile
0
20
40
60
80
100
120
0 10 20 30 45 60 90 120 125 130 135 140 150 165 180
Time (min)
Dru
g r
ele
ased
(%
)
Figure 3.17: Mean dissolution profile for coated 5% riboflavin pellets in simulated
gastric and intestinal fluids dissolution media.
3.9.7 Scanning electron microscopy
SEM enabled a detailed visualization of the empty yeast-glucan shells (Figure 3.18). It is
clear that the yeast-glucan shells have the form of small microparticles, and that the BSA
loaded shells have the same shape as the empty yeast-glucan shells.
SEM micrographs of the pellets showed that the pellets were spherical in shape and that
the surface of the pellets coated with Eudragit® 30LD-55 was smooth (Figure 3.18). A
cross-sectional view of the pellets revealed a rigid structure with low porosity and a
coating thickness of 38µm to obtain a gastroresistant effect (Figure 3.19).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 77
Figure 3.18: SEM pictures showing yeast shells (yeast-glucan), BSA loaded yeast-glucan
shells, uncoated BSA/yeast-glucan pellets, coated BSA/yeast-glucan pellets and cross-
section (half-sliced) coated BSA/yeast-glucan pellets.
Figure 3.19: SEM picture of a cross-section of a pellet coated with 30% (w/w)
Eudragit® 30LD-55, indicating coating thickness.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 78
3.9.8 Tablet hardness test
The hardness of a tablet refers to a measure of the crushing force exerted under
diametrical loading. The mean, minimum and maximum measured forces for the
representative tablets tested were 45N, 24N and 57N respectively. The obtained values
are considered acceptable in relation to this study since the minitablets were sufficiently
strong enough to withstand shear forces during friability testing and disintegrated within
the required time of 5 - 10 minutes since the hardness of a tablet affects its disintegration
time, the harder the tablet, the longer it takes to disintegrate.
3.9.9 Tablet friability test
Friability refers to the tendency for the tablets to form dusts or to break-up/off when
subjected to abrasion forces. It is the ability of the compressed tablet to avoid fracture and
breaking apart during transport.
BSA/yeast-glucan minitablets had a high mechanical strength, since friability values of
less than 1% was obtained (friability = 0.5%). The pharmacopeial limits consider a
maximum loss of 1 percent of the mass of the tablets tested to be acceptable. This low
friability indicates the tablets ability to withstand the shear forces when subjected to
mechanical shock or attrition.
3.9.10 Tablet disintegration test
The time taken for the minitablets to achieve complete disintegration was 5 - 10 minutes.
This showed that the composition of the formulation with the inclusion of lactose (which
is water soluble) and crospovidone (a superdisintegrant which is able to absorb large
amount of water and improve its penetration in the formulation and thus increase the
extent and rate of swelling of the tablets) are good choice as excipients for the
formulation of fast-disintegrating tablets leading to a fast release of the drug substance in
dissolution medium (Kristensen et al., 2002).
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 79
3.10 Conclusion
Yeast shells, extracted from S.cerevisiae have been shown to possess the ability to bind/
entrap antigens following tRNA/PEI cores formation by electrostatic interaction inside
yeast shells. With the purpose to serve as an oral vaccine delivery system, the yeast shells
were incorporated into pellets formulation prepared via extrusion/spheronization and
minitablets prepared by direct compression respectively.
Due to pellet disintegration, fast dissolution of “tracer pellets” containing riboflavin was
achieved (>80% drug release in 30 minutes) when using UNIPURE® EX starch as the
main excipient in pellet formulations prepared via the extrusion/spheronization technique.
Likewise, fast-disintegrating (<10 minutes) minitablets using crospovidone as
superdisintegrant and lactose could also be prepared by direct compression. The
disintegration time for both uncoated pellets (5 - 10 min) and minitablets (5 - 10 min) was
nearly the same, predicting similar bioavailability of the two developed types of dosage
forms when administered orally.
In conformity with data reported in the literature, this study has demonstrated that yeast-
glucan shells can bind/entrap antigens. The antigen-loaded yeast carriers when
formulated into enteric coated pellets and minitablets, can then be safely delivered to
macrophages and dendritic cells, thus, facilitating oral receptor-targeted (β-glucans
receptors) delivery of antigens.
Though in-vivo studies to evaluate the performance of the prepared delivery systems
(pellets and minitablets) are pending, the available data regarding the adjuvanticity of
yeast-glucan show that, it is a promising candidate for vaccine delivery system to induce
both mucosal and systemic immune responses.
D evelopm ent of m ulti-particulate form ulations for food and pharm aceutical applications 80
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