immobilization of biological material in...
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IMMOBILIZATION OF BIOLOGICAL MATERIAL IN BIOCOMPATIBLE
ALGINATE-POLYCATION ALGINATE MICROCAPSULE
A Project Report
Presented to
The Faculty of the Department of General Engineering
San Jose State University
In Partial Fulfillment
of the Requirements for the Degree
Master of Biomedical Devices Concentration
by
Arathi Asthi
Eric Chen
Kien Nguyen
December 2009
© 2009
Arathi Asthi
Eric Chen
Kien Nguyen
ALL RIGHTS RESERVED
ii
SAN JOSE STATE UNIVERSITY
The Undersigned Project Committee Approves the Project Titled
IMMOBILIZATION OF BIOLOGICAL MATERIAL IN BIOCOMPATIBLE
ALGINATE-POLYCATION ALGINATE MICROCAPSULE
by
Arathi Asthi
Eric Chen
Kien Nguyen
APPROVED FOR THE DEPARTMENT OF GENERAL ENGINEERING
__________________________________________________________________
Dr. Maryam Mobed-Miremadi, General Engineering Department Date
__________________________________________________________________
Dr. Mallika Keralapura, Electrical Engineering Department Date
__________________________________________________________________
Dr. Leonard Wesley, Computer Engineering Department Date
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ABSTRACT
IMMOBILIZATION OF BIOLOGICAL MATERIAL IN BIOCOMPATIBLE
ALGINATE-POLYCATION ALGINATE MICROCAPSULE
by
Arathi Asthi
Eric Chen
Kien Nguyen
Microencapsulation has been used extensively in oral drug delivery. The goal of
the project is to implement a robust microencapsulation methodology for delivery of
deficient enzymes to the GI tract. Literature review conducted on past and current
research resulted into an experimental approach using a Taguchi L9 design (4 factors, 3
levels). The effects of air flow rate (FA), concentration of polycation [cation], adsorption
time of polycation (Tads) and liquefaction time (Tliq) were analyzed. The microcapsules
were tested for average size, membrane strength and pH resistance. A first order linear
model linking microcapsule size to air flow rate was sufficient to predict the
microcapsule size over the range of the experimental matrix. Alginate Chitosan Alginate
(ACA) microcapsules showed higher resistance to pH degradation compared to Alginate-
Polylysine-Alginate (APA the gastro-intestinal transit simulation. A business model for
commercializing the technology is also discussed and market research has shown that the
oral drug delivery technology market is billion-dollar industry. A revenue model shows
that the company will start making profit by the end of the second year with 120% ROI.
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ACKNOWLEDGEMENT
First of all, we would like to express our gratitude and appreciation for Dr.
Maryam Mobed-Miremadi in General Engineering Department, San Jose State University
for sharing her enthusiasm and vast knowledge about the subject throughout the course of
the project. Without her support and guidance, the project would not have been possible.
We would like to Dr. Melanie McNeil, Professor in Material & Chemical
Engineering Department, and Dr. Mallika Keralapura, Assistant Professor in Electrical
Engineering Department, San Jose State University for being our technical readers.
We would like to thank Dr. Michael Jennings, Professor in Material & Chemical
Engineering Department, and Dr. Leonard Wesley, Associate Professor in Computer
Engineering Department, San Jose State University, for giving us invaluable guidance
during the course of the project.
Arathi Asthi
Eric Chen
Kien Nguyen
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TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION ...................................................................................... 1 1.1 Microencapsulation .................................................................................................. 1 1.2 Application of Microencapsulation .......................................................................... 2 1.3 Phenylketonuria ....................................................................................................... 2 1.4 Project objective ............................................................................................................ 5 1.5 Hypothesis..................................................................................................................... 5 CHAPTER 2: LITERATURE REVIEW ........................................................................... 6 2.1 Microencapsulation Methods ................................................................................... 6 2.2 Properties of alginate, poly-lysine and chitosan ...................................................... 9 2.3 Structure of a microcapsule ................................................................................... 13 2.4 Parameter effects on microencapsulation .............................................................. 14 CHAPTER 3: MATERIALS AND METHODS ............................................................. 16 3.1 Materials ..................................................................................................................... 16 3.2 Design of experiments ................................................................................................ 16 3.3 Preparation of APA microcapsules ............................................................................. 18 3.4 Preparation of Simulated Gastric Fluid (SGF) ............................................................ 18 3.5 Preparation of Simulated Intestinal Fluid (SIF) .......................................................... 18 CHAPTER 4: ECONOMIC JUSTIFICATION ............................................................... 19 4.1 Executive Summary ............................................................................................... 19 4.2 Solution and Value Proposition .................................................................................. 20 4.3 Market size .................................................................................................................. 21 4.4 Competitors ................................................................................................................. 22 4.5 Customers ................................................................................................................... 23 4.6 Cost ............................................................................................................................. 24 4.7 Price Point ................................................................................................................... 25 4.8 SWOT Assessment ..................................................................................................... 26 4.9 Investment Capital Requirement ................................................................................. 26 4.10 Personnel ................................................................................................................... 27 4.11 Strategic Alliances/Partners ...................................................................................... 27 4.12 P&L ........................................................................................................................... 28 4.13 Exit Strategy.............................................................................................................. 31 CHAPTER 5: PROJECT SCHEDULE ............................................................................ 32 CHAPTER 6: RESULTS and DISCUSSION................................................................... 34 6.1 Assessment of Microcapsule Size ............................................................................... 34 6.2 Assessment of the effect of airflow, poly-cation concentration, adsorption time, and liquefaction time on the size and signal/noise ratio of the microcapsule ......................... 39 6.3 Assessment of leakage ................................................................................................ 43 6.4 Assessment of pH resistance ....................................................................................... 45 6.5 Prediction Model ......................................................................................................... 48 CHAPTER 7: CONCLUSION ......................................................................................... 51 REFERENCES ................................................................................................................. 52 APENDIX A ..................................................................................................................... 55
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LIST OF FIGURES
Figure 1. Schematic Diagram of the Co-axial Needle Assembly. .................................................. 9 Figure 2. Schematic Diagram of the Preparation of Alginate Poly-lysine Microcapsule
(Yeo et al., 2001) .................................................................................................................... 9 Figure 3. Structure of Alginic Acid. A segment of an alginate molecule is comprised of
two G and two M monomers. ............................................................................................... 11 Figure 4. Structure of Poly L-lysine. ............................................................................................ 11 Figure 5. Structure of Chitosan. A segment of a chitosan molecule includes two D-
glucosamines and one N-acetyl-D-glucosamine. .................................................................. 12 Figure 6. Structure of Alginate Poly-lysine Alginate Microcapsule (Vos et al., 2008). ............... 14 Figure 7 The Worldwide Oral Drug Delivery Market .................................................................. 22 Figure 8. Breakeven Analysis ....................................................................................................... 29 Figure 9. Funding Profile in the First Year ................................................................................... 30 Figure 10. Accumulative Funding in the First Year. ................................................................... 30 Figure 11. Microcapsule with mean size of 732 µm (FL-0.3 ml/min, FA – 2.6 L/min) .............. 35 Figure 12. Microcapsule with mean size of 633 µm (FL-0.3 ml/min, FA – 2.9 L/min) ................ 35 Figure 13. Microcapsule with mean size of 543 µm (FL-0.3 ml/min, FA – 3.2 L/min) ................ 36 Figure 14 Frequency distribution of 60 microcapsules (FL- 0.3 ml/min and FA- 2.6 L/min) ....... 37 Figure 15. Frequency distribution of 60 microcapsules (FL- 0.3 ml/min and FA- 2.9
L/min) ................................................................................................................................... 38 Figure 16. Frequency distribution of 60 microcapsules (FL- 0.3 ml/min and FA- 3.2
L/min) ................................................................................................................................... 39 Figure 17. Effect of air flow rate poly cation concentration, adsorption time, and
liquefaction time on microcapsule size ................................................................................. 42 Figure 18. Effect of air flow rate poly cation concentration, adsorption time, and
liquefaction time on microcapsule signal/noise ratio ............................................................ 43 Figure 19. Blue dextran calibration curve .................................................................................... 44 Figure 20. Blue dextran leakage as a measure of microcapsule strength ..................................... 45 Figure 21. ACA microcapsules after 2 hr incubation in SGF ....................................................... 46 Figure 22 APA microcapsules after 2 hr incubation in SGF ........................................................ 47 Figure 23 Swollen ACA Microcapsules after 2 hrs Incubation in SGF followed by 18 hrs
Incubation in SIF................................................................................................................... 47 Figure 24. Dissolved APA Microcapsules after 2 hrs Incubation in SGF followed by 18
hrs Incubation in SIF ............................................................................................................. 48 Figure 25. First order linear model plot to predict the size of the microcapsule in terms of
air flow rate. .......................................................................................................................... 50
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LIST OF TABLES
Table 1. Different types of Hyperphenylalaninemia (Stevens, 2007). ........................................... 4 Table 2. Incidence of PKU in Certain Countries (Lindner, 2007; Visakorpi et al., 2008). ............ 4 Table 3. Variables and Levels. ...................................................................................................... 17 Table 4. Optimizations Type by Response ................................................................................... 17 Table 5. L9 (4 variables, 3 levels) Taguchi Matrix for the Experiment. ....................................... 17 Table 6. The Worldwide Oral Drug Delivery Market (All figures are in billions of
dollars) .................................................................................................................................. 21 Table 7. Leading Drug Companies. .............................................................................................. 24 Table 8. Calculation for Total Cost. .............................................................................................. 25 Table 9. SWOT Analysis .............................................................................................................. 26 Table 10. Calculation of Return of Investment. ............................................................................ 28 Table 11. Calculation of Normalized Accumulative Cost Driver ................................................. 29 Table 12. Project schedule ............................................................................................................ 32 Table 13. The Gantt chart of the project schedule. ....................................................................... 33 Table 14. Summary of Taguchi Analysis of Microcapasule Size Characterustic using the
Nominal the Best Optimization ............................................................................................ 41
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LIST OF ABBREVIATIONS
ACA Alginate chitosan alginate APA Alginate poly L-lysine alginate ERT enzyme replacement therapy GI Gastrointestinal tract IEM Inborn errors of metabolism PAH Phenylalanine hydroxylase Phe Phenylalanine PKU Phenylketonuria PLL Poly L-lysine RESS Rapid expansion of supercritical solutions SAS Supercritical antisolvent crystallization SGF Simulated gastric fluid SIF Simulated intestinal fluid S/N Signal to noise ratio OFAT One-Factor-at-A-Time FA Air flow rate Tads Adsorption time of poly-cation Tliq Liquefaction time
CHAPTER 1: INTRODUCTION
1.1 Microencapsulation
Microencapsulation is a process by which a continuous film of polymeric material
is coated on tiny droplets or particles of liquid or solid material. In other words, a
microcapsule is a small sphere with a uniform surrounding wall. The material inside the
microcapsule is called as the core, internal phase, or fill, while the wall is sometimes
referred to as a shell, coating or membrane (Kamyshny et al., 2006). Most of the
microcapsules are very small spheres whose diameter ranges from a few micrometers to a
few millimeters (Torrado et al., 2008). Bioencapsulation is the microencapsulation of a
biologically active compound as a core material that is allowed to release at a certain rate
(Socaciu, 2007).
Microencapsulation technology, hence, helps to endow biological materials with
many superior features by:
Allowing a high concentration of biological materials to reach the target.
Boosting the optical, thermal or chemical stability of the core compounds.
Extending the shelf life of active materials as a result of the higher stability.
Managing the discharge of active components from the core through the shell.
Mixing incompatible constituents in the core.
Protecting the biologically reactive materials such as enzymes against the harsh
environment of the body (Kamyshny et al., 2006).
Microencapsulation controls timely release of a biological material such as an
enzyme, allowing the enzyme to degrade slowly over a period of time. The enzyme is
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protected inside the microcapsule from the surrounding environment until the designated
time for the enzyme to be released. The material is then released by various mechanisms
such as rupturing, diffusion, melting or dissolution. Oral delivery of enzyme is
considered more efficient than injection in treating many diseases (Lindner, 2007;
Visakorpi et al. 2008).
1.2 Application of Microencapsulation
Microencapsulation technology can be applied to many different applications in
food, feed, electronics, graphics and printing, photography, textiles, waste treatment,
agriculture, chemical industry, pharmaceuticals, biotechnology, household and personal
care, human and veterinary medicine.
One of the potential applications of microencapsulation technology is enzyme
replacement therapy for treating Inborn Errors of Metabolism (IEM). The majority of
IEMs are due to mutations of single genes that code for enzymes that assist in conversion
of various substances into other products. The disorders can create problems when
unwanted substances that are toxic, interfere with normal function, or reduce the ability
to synthesize essential compounds being accumulated. Phenylketonuria (PKU) is a well-
documented case example of IEMs.
1.3 Phenylketonuria
Phenylketonuria is a genetic disorder in which a newborn is deficient in or has
very low levels of the enzyme phenylalanine hydroxylase (PAH). The enzyme PAH is
necessary to hydroxylate the amino acid phenylalanine (Phe) to form tyrosine. With the
absence of PAH, a considerable volume of Phe accumulates in the blood that, if not
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controlled, could lead to damage of brain tissue, causing mental retardation and central
nervous system problems (Visakorpi et al., 2008).
The main treatment currently available for PKU is a lifelong, special reduced-
protein diet, which helps to prevent phenylalanine from building up in the body. Many
protein foods contain about 5% Phe; therefore, the diet that is designed for these patients
should be strictly adhered and also adequately supplemented with vitamins and nutrients
for their proper growth and development. The different types of hyperphenylalaninemia
are shown in Table 1. BioMarin, a pharmaceutical company in Novato, California, is in
the process of developing a new drug for treatment of PKU. The drug is still in a clinical
trial phase and hasn’t been proven safe and effective yet. The other ideal approach for
treatment of PKU would be oral administration of the missing enzyme by
microencapsulating it. This approach is called enzyme replacement therapy (ERT)
(Lindner, 2007).
Phenylketonuria is one of the most common inherited disorders in the nation: 1
out of 15,000 to 1 out of 19,000 US newborns is affected with PKU due to the presence
of two mutant genes from the enzyme phenylalanine hydroxylase (PAH) (Visakorpi et
al., 2008). Table 2 lists the incidence of PKU affecting newborns in other countries.
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Table 1. Different types of Hyperphenylalaninemia (Stevens, 2007).
Blood Level of PHE Diagnosis Treatment (mg/dL) Micromoles/Liter
<4 <240 Normal None
4 – 10 240-600 Mild Hyperphe
A low PHE diet is usually not prescribed during childhood.
A low PHE diet may be needed during pregnancy.
10.1 - 19.9 606-1194 Atypical PKU or Hyperphe Variant
Requires a low PHE diet.
Women must have a low PHE diet before and during pregnancy.
20 and higher 1200 or higher Classical PKU
A low PHE diet is needed to prevent mental retardation.
Women must have a low PHE diet before and during pregnancy.
Table 2. Incidence of PKU in Certain Countries (Lindner, 2007; Visakorpi et al., 2008).
Incidence Statistics
Ireland 1/4500 births
Finland 1/100,000 births
Caucasians in United States 1/8,000 birth
Blacks in United States 1/50,000 birth
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Phenylketonuria is rare, but considered as a serious problem, which results in a
significant market potential for treatment of PKU using the microencapsulated enzyme
PAH. Traditional treatment methods (diet restriction) do not effectively control the
levels of Phe in the bloodstream, nor there any drugs readily available that could treat this
disease. Therefore, it is a necessity to develop a model membrane that can protectively
carry the deficient enzyme to the desired location in order to breakdown phenylalanine to
form tyrosine, and control the accumulation of Phe in the bloodstream.
1.4 Project objective
The project has two main objectives:
To optimize the microencapsulation conditions in order to reproducibly
control the average size of the microcapsules (d=500 µm + 200 µm) and
prevent leakage by controlling membrane strength.
Test the stability of microcapsules under various pH conditions to simulate
the oral administration path in the GI (gastro-intestinal tract).
1.5 Hypothesis
It is possible to prepare a homogenous batch of microcapsules that is:
Minimally affected by the physiologic pH between 1 and 8, and
Mechanically stable so that the immobilized enzyme does not leak out.
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CHAPTER 2: LITERATURE REVIEW
2.1 Microencapsulation Methods
Several types of microencapsulation methods have been used such as solvent
evaporation or extraction, phase separation, spray drying, ionotropic gelation or
polyelectrolyte complexation, interfacial polymerization, supercritical fluid precipitation
(Yeo et al., 2001).
Solvent evaporation or extraction is used for making microcapsules loaded with
different drugs, especially for hydrophobic drugs. Emulsification of oil/water, oil/oil and
water/oil/water is performed for encapsulating the peptide and protein drugs. It is good
for delivery of small molecule drugs with low aqueous solubility, faster release of loaded
drugs. However, the low drug encapsulation efficiency into microspheres drives the cost
up. Moreover, the method requires the use of toxic organic solvents compromising the
kinetics of release.
Phase Separation includes three steps: phase separation of the coating polymer,
adsorption of coacervate droplets, and solidification of the microcapsules. This method
minimizes the loss of water-soluble drugs to water phase and results in high
encapsulation efficiency. In addition, it allows efficient control of particle size with a
narrower size distribution by simply varying the component variables. The limitations
include tendency of microspheres to aggregate, difficulty of mass production, toxicity of
residual solvents.
Spray drying is used in the pharmaceutical, food, and biochemical industries.
Encapsulation is one of the main applications in the field. Polymer and drug are
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dissolved in the solvent. Drug and polymer solution are then emulsified. Afterward, the
sprayed mixture turns into solid microspheres. General applicability is one of the major
advantages of this method. It is also very useful in encapsulating heat sensitive drugs.
However, material can be lost during the process because micro particles can stick to the
wall of the drying chamber. Spray drying can also lead to change of polymorphism of
spray dried drugs.
Interfacial polymerization is defined as the polymerization of reactive monomers
on the surface of a droplet or particle. Two reactive monomers are dissolved in solvents
and mixed to form oil/water emulsion. Reaction takes place to form a polymeric
membrane from the monomers. The method is ideal for preparing insulin nano particles
and enzyme encapsulation; however, there are some serious problems associated with this
method. Firstly, the proteins or enzymes can be inactivated at the large water/oil
interface. Secondly, the polymerization reaction can alter the biological activity of the
proteins. Thirdly, it is very difficult to control the rate of polymerization. At the
supercritical state the temperature and pressure of the fluid are higher than the critical
point. There are two ways for supercritical fluids to form particles: rapid expansion of
supercritical solutions (RESS), and supercritical anti solvent crystallization (SAS). Drug
and polymer are dissolved in supercritical fluid at high pressure during RESS and thus
reducing the solvent’s density in order to form precipitation. Supercritical fluid is used as
an anti-solvent in SAS. Supercritical fluids are considered as relatively non-toxic, non
flammable, inexpensive, and environmentally acceptable. However, it is not applicable
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for high polymers because of the low solubility in supercritical fluid. It is also difficult to
control and predict the precipitate.
Ionotropic gelation/polyelectrolyte complexation can be also referred as the
atomization method. It is based on the ability of polyelectrolytes to crosslink for forming
hydrogels. Microspheres can be created by atomizing the polyanionic alginate solution
that contains cells or drugs into aqueous CaCl2 solution. An ionically -cross linked
alginate can be formed by diffusing calcium ions into the alginate drops. Mechanical
strength of the hydrogel can be increased by adding a polyelectrolyte complexation with
oppositely charged polyelectrolytes. This reaction can also help to create a permeability
barrier. Addition of polycation can allow the polyelectrolyte complex membrane to form
on the alginate beads surface. A coaxial needle assembly is used to atomize the droplets.
The schematic diagram of the needle assembly is shown in the Figure 1. A vibration
system or air atomizer can be used to extrude the alginate solution if smaller droplets are
desired. The calcium ions form microgel droplets by crosslinking the droplets of sodium
alginate in contact, then further cross linking with poly L-lysine to create a membrane on
the droplets. The experimental set up of the procedure is shown in the Figure 2.
The key advantage of this method is that organic solvents or elevated
temperatures are not needed to encapsulate the proteins. The system is also considered as
simple, fast, and inexpensive. The biggest problem of the method is that the protein
release rate is hard to control for a long period of time from the membrane. In order to
control the release rate effectively, a certain type of dense membrane should be
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incorporated. Optimizing the adsorption conditions and properties of polycationic
electrolytes can also control the permeability of the membrane.
Figure 1. Schematic Diagram of the Co-axial Needle Assembly.
Figure 2. Schematic Diagram of the Preparation of Alginate Poly-lysine Microcapsule (Yeo et al., 2001)
2.2 Properties of alginate, poly-lysine and chitosan
Immobilization of biological materials, a process by which biological material are
deposited on a substrate, commonly occurs in nature such as the forming of a film by
bacteria on soil particles, plants or on animal tissues such as intestine and rumen. People
used coating of bacteria in the manufacturing of vinegar and leaching of mineral oil ores
9
by sulphur oxidizing bacteria. The driving force behind these applications is the catalytic
action of the enzyme secreted by the microorganism. Since the enzymes have specific
catalytic activity and high performance under mild physiological conditions, they have
become increasingly important in fermentation. Being used in aqueous systems, enzymes
can be in the free form or in insoluble form. When immobilized on a solid substrate, the
enzyme results in operational stability and enhanced activity (Phillips et al., 1988). Many
biomaterials have been used to immobilize enzymes. For example, carbohydrates include
cellulose, agar, agarose, or k-Carrageenan; proteins include collagen, gelatin or albumin;
and synthetic polymers include polyacrylamide or polyurethane (Xiajun et al., 1994).
Among the carbohydrates used as carriers for enzyme or cell immobilization is
alginate, the widely used biomaterial that is extracted from the giant kelp (Macrocystis
pyrifera). Alginic acid is an unbranched binary copolymer of D-mannuronate (M) and L-
guluronate (G) (Figure 2). Two G monomers form bonding with divalent ions such as
barium or calcium, or with bi-functional molecules such as methyl ester L-Lysine or
polyethylene glycol. In the presence of these molecules, alginate, the ester or salt of
alginic acid, will be covalently cross-linked, forming a three-dimensional network or gel.
The gelling and mechanical properties of the alginate gel will depend on the ratio of G
and M in the alginate and on the distribution of G and M in the polysaccharide. Alginate
gel does not promote protein adsorption; therefore, the polyanionic alginate beads might
be coated with polycations such as poly-L-lysine (PLL) or chitosan to establish selective
permeability and maintain the capsule durability and biocompatibility in vivo (Riddle et
al., 2004; Melvic et al., 2004).
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Figure 3. Structure of Alginic Acid. A segment of an alginate molecule is comprised of two G and two M monomers. (http://www.lsbu.ac.uk/water/images/hyalg.gif)
The protective layer of an alginate gel can be formed from a poly-cationic
compound such as poly L-lysine, a polymer of L-lysine, an essential amino acid that
cannot be synthesized in the body. However, poly L-lysine is found to trigger an immune
response from the body; therefore, a poly L-lysine coated alginate microcapsule needs
another coating to make it resistant to the immune attack. In 1980, the use of APA
capsule to encapsulate cells by Lim and Sun initiated an intense interest in the
microencapsulation area. However, initial clinical trials showed that APA capsules
exhibited poor mechanical stability and biocompatibility. Further research and
experiments showed that capsule, membrane, gel quality and performance depended on
alginate microstructures, molecular weight of poly l-lysine, and preparation conditions.
Figure 4. Structure of Poly L-lysine. (http://commons.wikimedia.org/wiki/File:L-Lysine.png)
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Chitosan is another polycationic polymer that can be used as the protective
coating of an alginate bead. Chitosan can form gels by cross-linking with glutaraldehyde
and degrade via enzymatic hydrolysis. Chitosan, an unbranched copolymer of D-
glucosamine and N-acetyl-D-glucosamine (Figure 3) is derived from chitin, which is
plentiful in the exoskeletons or hard shells of crustaceans. Chitosan, like alginate, has
been used in many biological applications such as wound dressings or drug delivery
systems. Since chitosan is not as biocompatible as alginate, it is not used in injection
drug delivery systems as an immobilizing material. However, in oral drug delivery
systems, chitosan might show greater potential than other polycationic compounds.
Consequently, ACA microcapsules have not been extensively studied as APA
microcapsules (Riddle et al., 2004).
Figure 5. Structure of Chitosan. A segment of a chitosan molecule includes two D-glucosamines and one N-acetyl-D-glucosamine.
(http://commons.wikimedia.org/wiki/File:Chitosan_Synthese.svg)
In oral drug delivery microcapsules must pass through the GI tract before the
target destination is reached. During its travel, the microcapsule is exposed to different
acid levels since the pH of the stomach and GI tract can vary from 1 to 8 (pH 1-3 in the
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stomach, pH 6-6.5 in the duodenum, pH 5.5-7 in large intestine); therefore, it is necessary
to develop a membrane coating than can withstand various ranges of pH and also be
mechanically strong. Alginate-chitosan-alginate (ACA) microcapsules have been shown
to be mechanically stable in a larger pH range than alginate poly L-lysine alginate (APA)
microcapsules, especially in the range of pH 2-5 (Riddle et al., 2004).
2.3 Structure of a microcapsule
The alginate-polycation-alginate microcapsule contains a core and two additional
layers:
The core of the capsule: Functions to immobilize the enzyme from the
bulk calcium alginate.
The polycationic layer: Functions to stabilize and strengthen the core and
also control the core permeability.
The polyanionic layer: Functions to control the permeability and
neutralize the charge on the polycationic layer to avoid any adherence to
the capsule (Bruheim et al., 1996). A diagram of an alginate poly L-lysine
alginate (APA) microcapsule is shown in Figure 3.
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Figure 6. Structure of Alginate Poly-lysine Alginate Microcapsule (Vos et al., 2008).
2.4 Parameter effects on microencapsulation
Forming a consistent microcapsule involves controlling several parameters like
viscosity, air flow rate, adsorption time, molecular weight, etc. The literature search
resulted in the following operating range for the variables considered for making a
microcapsule of size 500 μm + 200 μm:
Thu et al. (1996) studies showed that the capsule formation was dependent
on the concentration, composition, molecular weight of the polymer being used and also
the exposure time to the poly-lysine. Their studies also showed that by varying the
concentration of the poly-lysine and their exposure time, the strength of the membrane
could be varied.
14
Gugerli et al. (2002) characterized the APA microcapsules using analytical
methods. His studies reported that the molecular weight of PLL was a key determinant
for the resistance and permeability of the capsule.
Paul et al. (2008) reviewed the association of the molecular weight to the
viscosity and rheological properties. It was necessary to “optimize capsule size, size
distribution, mechanical resistance, permeability and membrane thickness in terms of
time, concentration and molecular weight” (Gugerli et al. 2002).
Thu et al. (1995) suggested the adsorption time for the polycation to be
between 1-20 minutes for proper coating of the polycation on the alginate beads.
However for chitosan coating, Lin et al. (2008) controlled the flow rate of
alginate to 30 ml/min and used 0.5% w/v concentration of chitosan. The beads were
shaken for 30 mins.
Tatiana et al. (2008) tested the different airflow rates (5 L/min, 10 L/min
and 15 L/min) for alginate bead formation.
15
CHAPTER 3: MATERIALS AND METHODS
3.1 Materials All chemicals used to make the microcapsules were purchased from Sigma
Aldrich:
Medium molecular weight sodium-alginate (A2033),
Low molecular weight sodium-alginate (A0682),
Polylysine-hydrobromide 20,000<MW<30,000 (P81333),
Hemoglobin powder (H2500).
The 16 G and 24 G stainless steel needles in the atomizer assembly were
procured from Popper and Sons.
Trypsinase
A UV-VIS spectrophotometer (HP8453)
NaCl, CaCl2, sodium citrate, KH2PO4, NaOH, pepsin, pankreatin
3.2 Design of experiments
Based on literature search, we decide to limit our experiment to 5 variables.
Instead of using factorial design for the first pass optimization, we are going to use a L9
(4 variables, 3 levels) Taguchi design technique to find the optimal range of these 4
parameters for making mechanically strong microcapsules with a diameter of 500 μm +
200 μm. The Taguchi design parameters, variable levels and optimization types, and
actual value for each parameter in the 9 runs are shown in Tables 3-5 respectively. The
experiment summarized in Table 5 will be performed for two types of polycations, poly
L-lysine and chitosan used for coating the alginate beads.
16
Table 3. Variables and Levels.
No Variables Levels 1 Flow rate of air: 2.6-3.2 L/min 2.6,2.9,3.2 2 Concentration of Poly-cation: 0.01 w%/v%-0.03
w%/v% 0.01,0.02,0.03 3 Adsorption time of Poly-cation: 5min-25 min 5,15,25 4 Sodium Citrate liquefaction time 2-4 min 2,3,4
Table 4. Optimizations Type by Response
Responses Type of Optimization Diameter Nominal the best Strength Bigger the Better pH resistance Bigger the Better
Table 5. L9 (4 variables, 3 levels) Taguchi Matrix for the Experiment.
Runs Air Flow rate (FA)
(L/min)
Concentration of Polycation
[Cation] (w%/v%)
Absorption time of
Polycation (Tads)(min)
Liquefaction Time (Tliq)
(min) 1 18 0.01 5 2 2 18 0.02 15 3 3 18 0.03 25 4
4 20 0.01 15 4 5 20 0.02 25 2 6 20 0.03 5 3
7 22 0.01 25 3 8 22 0.02 5 4 9 22 0.03 15 2
Two other potential variables are the molecular weight of the alginate and the
dispense tip material. They are not considered part of this initial experimental matrix.
The concentration of the alginate can be modulated to compensate for the viscosity
dependence of the weight average molecular weight. The tip material is hydrophilic
17
material such as stainless steel compatible with the hydrophilicity of the biological
compounds.
3.3 Preparation of APA microcapsules
1.5% medium viscosity sodium alginate (µ=500 cP, σ= 45 mN/m) mixed with
blue dextran aliquoted in 2 ml batches was atomized through the concentric needle
assembly depicted in Figure 1. After the initial one factor at a time (OFAT) screening
experiments the flow rate of liquid (FL) was fixed at 0.3 ml/min and the air flow rate (FA)
was varied to produce microcapsules of different sizes. The atomized alginate beads
were dispensed into 1.5 M CaCl2. After 6 min the beads were centrifuged and washed
twice with 0.9% NaCl. The washed beads were then coated with poly-lysine for various
adsorption times (Tads). The beads were washed with saline twice and coated with 0.1%
medium viscosity sodium alginate for 4 minutes followed by two saline washes. The
resulting APA beads were suspended in 1.5% 55mM sodium citrate for various core
liquefaction times.
3.4 Preparation of Simulated Gastric Fluid (SGF) SGF was prepared according to the United States pharmacopeia (USP 24) to
simulate the microcapsules in vivo. 0.32g pepsin, 0.7ml of 37% Hcl, and 0.2g of NaCl
was added to 100 ml of DI water and the pH was adjusted to 1.7
3.5 Preparation of Simulated Intestinal Fluid (SIF) SIF was prepared according to the United States pharmacopeia (USP 24) to
simulate the microcapsules in vivo. 1.0 g pankreatin, 0.680g of KH2PO4, and 0.062g of
NaOH was added to 100 ml of DI water and the pH was adjusted to 7.5
18
CHAPTER 4: ECONOMIC JUSTIFICATION
The economic justification will give an overview of our microencapsulation
technology called MicrocapTech, value proposition, market size, customers, and
competitors in the oral drug delivery technology market, SWOT analysis, strategic
alliance and exit strategy. The justification will also provide a discussion on the financial
aspects of the project in terms of cost, price point, capital investment, profit/loss
calculation, break-point analysis, and return on investment. This part will also include a
control measure for budgeting during the project.
4.1 Executive Summary
The market of oral drug technologies is usually integrated into a larger market-
the oral drug delivery market that includes the sale of oral drug technologies and the sale
of pharmaceuticals from these technologies. Some diseases require the medication to be
absorbed into the bloodstream via injection or pills for the treatment to be effective, other
diseases can be treated by administration of the medication via the preferred oral route to
the GI tract. The pharmacological activities of the medication will occur outside the
bloodstream, eliminating the need for the
BioSphere Ltd. is a R&D company that develops an innovative drug delivery
technology called MicrocapTech, which implements an oral drug delivery system in
treatment of inborn errors of metabolism such as PKU. MicrocapTech is a
microencapsulation technology used to make Alginate-polycation-Alginate
microcapsules, which deliver deficient enzyme to the GI tract. This economical and
innovative approach to treat IEM distinguishes itself from other oral drug delivery
19
technologies employed by its numerous customers and competitors in the market. The
cost for operating the company is $140,000 in the first year, and $90,000 for subsequent
years; therefore, the company will need $140,000 capital investment in the first year to
get the project started and the company will start to make profit by the end of the second
year by licensing out the technology to its potential customers at the fee of $200,000 per
year on an exclusive basis. As the business expands, the company might work with its
strategic partners on other microencapsulation technologies in treatment of IEM.
Alternatively, the company might sell its product and exit the market, or merge with its
competitors or customers.
4.2 Solution and Value Proposition
Microencapsulation has been largely used in pharmaceutical and medical fields to
immobilize drugs and enzymes (Klein, 1985); however, the technology has not yet been
utilized in treatment of genetic disorders such as PKU. MicrocapTech is our
microencapsulation technology used to make Alginate-polycation-Alginate
microcapsules, which will deliver the deficient enzyme, PAH in this case, to the GI tract.
Upon reaching the GI tract, the enzyme will catalyze the conversion of Phenylalanine to
tyrosine, hence, lowering the level of Phenylalanine entering the blood stream.
As a consequence, MicrocapTech is an innovative and extensive approach to
treatment of genetic disorders. For example, PAH-immobilized microcapsules are the
cure for all PKU patients while Kuvan, manufactured by BioMarin, is only effective for
30-50% PKU patients (BioMarin, 2008). BioMarin use a synthetic analog of
Tetrahydrobiopterin (BH4), a cofactor of PAH enzyme. The cofactor BH4 is intended to
20
fix the impaired PAH and boost up PAH level in all PKU patients; however, it is only
effective in 30-50% PKU patients. Our innovative solution MicrocapTech will be the
comprehensive approach to deliver deficient enzymes in patients inflicted by metabolic
inborn errors such as PKU.
4.3 Market size
PKU is a rare condition that only happens to 1 out of 15,000 to 1 out of 19,000
babies born in the US. The revenue generated by our competitor, BioMarin, due to
Kuvan alone, was $400,000 in 2007. However, our product is MicrocapTech; therefore,
it is necessary to mention the market size of drug delivery technology.
According to Marketresearch.com Inc., the market for oral drug delivery includes
the revenue generated from licensing the drug technology and the revenue earned from
selling the pharmaceutical that uses the technology. The total market for oral drug
delivery, the market for the delivery technology and the market for the pharmaceutical
using the delivery technology are shown in Table 6 and 7.
Table 6. The Worldwide Oral Drug Delivery Market (All figures are in billions of dollars)
Year Oral Delivery Technology
Pharmaceuticals using the technology
Total market (in Billions)
2006 3.3 32.3 35.6 2007 3.5 35.5 39 2008 3.2 39.8 43 2009 3.5 43.9 47.4 2010 3.7 48.6 52.3 2011 4 53.9 57.9 2012 4.3 59.9 64.2 2013 5 66.3 71.3
21
Worldwide Oral Drug Delivery Market
3.3 3.5 3.2 3.5 3.7 4 4.3 5
32.3 35.539.8 43.9
48.653.9
59.966.3
010
2030
4050
6070
2006
2007
2008
2009
2010
2011
2012
2013
Year
Dol
lars
(Bill
ion)
Technology Pharmaceuticals
Figure 7 The Worldwide Oral Drug Delivery Market
4.4 Competitors
BioMarin is the only company that aims to provide treatment for IEMs such as
PKU in the United States, but they do not employ microencapsulation technology. There
are several pharmaceutical companies that use microencapsulation to implement their
oral drug delivery system but none of them use microencapsulation technology in
treatment of genetic disorders like PKU. Most of them use microencapsulation as taste
masking technique. These companies are considered potential competitors and some of
their microencapsulation technologies are worth mention here.
MicroCaps ® is a patented microencapsulation technology, developed by Eurand,
a specialty pharmaceutical company in the Netherlands. The company uses the
coacervation method to coat drug particles in a polymeric membrane whose thickness and
porosity will shield the unpleasant taste of the drug and allow the drug to release in a
controlled fashion.
22
DuraSolv ® and OralSolv ® are two microencapsulation technologies developed
by CIMA Laboratories, an independent business unit of Cephalon company, a U.S.
biopharmaceutical company headquartered in Philadelphia, Pennsylvania. The drug-
containing microcapsules are kept in tablets, which, upon swallowing, will dissolve and
release the microcapsules as a slurry or suspension. The technologies are also used for
the purpose of taste masking and drug release control.
Another microencapsulation technology used for the same purpose is
Micromask® developed by Particle Dynamics Inc., a business division of KV
pharmaceutical, located in St. Louis, Missouri. The microcapsule-containing tablets will
quickly dissolve, releasing the drug encapsulated particles into the mouth without the
need of water.
4.5 Customers
Our customers are any pharmaceutical companies that need a new enzyme
delivery technology in their oral drug delivery portfolio. The potential competitors
mentioned above could be our primary customers for our product MirocapTech. Table 7
lists more customers in oral drug delivery technology market, together with some of their
proprietary technologies, and revenue in 2007.
23
Table 7. Leading Drug Companies.
Company y s
Proprietary oral delivertechnologie
Revenue in 2008 ($ millions)
ALZA (division of J&J)
OROS NA
Biovail www.biovail.com
Ceform, Dimatrix, Macrocap 757,178
CIMA www.cimalabs.com
OraSolv, DuraSolv 1,974
Eurand www.eurand.com
MicroCap 100,000
Elan www.elandrugdelivery.com AS
NanoCrystal, CODAS, SOD 300
Emisphere ere.comwww.emisph
Protenoid Oral Drug Delivery 0.251
Ethypharm
.comwww.ethypharm Oral modified released, taste-
203.7
masked and orodispersible
KV Pharmaceutical www.kvpharma.com
FlavorTech, Micromask, 601,897Liquette, MeterRelease,
Labopharm www.labopharm.com Contramid, Polymeric Nano- 22,014 Delivery Penwest www.penwest.com
Rx, ProSolv, Geninex 8,543
TIME
SkyePharma www.skyepharma.com
GEOMATRIX 99.3
4.6 Cost
24
The cost for developing the microencapsulation technology includes the variable
ixed cost. The fixed cost includes the cost of renting an office and paying
salaries
the
2009 2010 2011 2012 2013
cost and f
. The variable cost includes the cost of renting laboratory equipment, purchasing
computers and software in the 1st year. Table 8 shows the details and calculation for
total cost.
Table 8. Calculation for Total Cost.
Year
Fixed cost ($) 90,000 90,000 90,000 90,000 90,000
Variable cost ($) 50,000 0 0 0 0
Total cost ($) 140,000 90,000 90,000 90,000 90,000
4.7 Price Point
The product or the design is priced low at first in order to attract more
ng is a very important factor in controlling the supply and demand. If the
initial p and more
ion
be
customers. Prici
rice of the product/design is lower, then there will be a higher demand
customers would be willing to pay for it. The price is calculated according to several
factors: 1) the supply and demand curve, 2) market analysis, 3) cost and expenses, 4)
development schedule of our products. We are looking to license the microencapsulat
technology to the customers instead of the pharmaceutical products. The price of the
design or licensing should be $200,000 per year. At the same time, our company will
apply for a patent in other countries other than US. At that time the licensing fee will
calculated according to the currency from each country.
25
4.8 SWOT Assessment
SWOT Assessment is a very helpful tool in evaluating the strengths, weaknesses,
in a project or company. It provides a discipline in measuring a
compan
• The use of natural components is
t “go-green”
aterials and
• The technology MicrocapTech is
subject to strict regulations.
opportunities, and threats
y and its other issues. Strengths and weaknesses are considered as internal
factors, while opportunities and threats are considered as external factors.
Table 9. SWOT Analysis
Strength Weakness
aligned with the curren
movement.
• The per-patient price is low due to
low cost of m
manufacturing process.
• The total cost of R&D is high.
Opportunities • Market is expanding due to more
genetic disorders being discovered.
Threats • Other more natural approaches such
as dietary supplement of large neutral
t
ers
• MicrocapTech will provide a com
vehicle to deliver enzymes other than
mon
amino acids or low phenylalanine die
therapy are used to treat PKU.
• Gene therapy might be an alternative
approach to treat genetic disord
PAH to GI tract.
4.9 Investment Capital Requirement
26
BioSphere is a R&D consultancy firm that is in search of $ 140,000 in the first
year of
e is a R&D company that was established in January 2009 to develop a
microe
U. The
ility
ncludes 3 graduate students. Each of us has a
broad k
4.11 Strategic Alliances/Partners
its operation. The funding that we receive from the investors will be used to
renting necessary laboratory equipments and facility, implementing an information
technology infrastructure that helps in product design and development process, and
implementing sales and marketing plans. Since it takes at least one year to develop
MicrocapTech, the company is expected to become profitable by the second year.
4.10 Personnel
BioSpher
ncapsulation technology. The technology is aimed to provide a common
framework for oral enzyme delivery systems in treating IEM diseases such as PK
company starts with a workforce of 3 people. It might grow towards a larger workforce
as the business expands. The company has decided the legal form of organizing the
company as a Limited Liability Company (LLC). This form of organization has
characteristics of both a corporation and a partnership. The owner has limited liab
for the actions and debts of the company.
The R&D staff of BioSphere Ltd. i
nowledge in different fields-biotechnology, biomedical devices, and healthcare.
We are familiar with laboratory instrumentations and equipment, as well as different
experimental design techniques. We collaborate with universities and industrial
consultants in our current project.
27
BioSphere Ltd. searches for a new microencapsulation technology used in
medica tical
res
r IEM,
le 10 shows the loss/profit statement for the next 5 years. From the table, the
Total cost
Retu
11 2012 2013
l applications; therefore, the company does not manufacture any pharmaceu
products. Since the company is a small startup company, it should form a strategic
alliance with an existing company such as BioMarin Pharmaceutical Inc., which sha
the same mission. Since the company’s technology is complement to BioMarin’s
technology, this alliance might help BioMarin to diversify their treatment option fo
increasing the effectiveness of their treatment that they are facing right now. This
alliance will also help us to establish our new technology in the IEM market.
4.12 P&L
Tab
plot of cost versus revenue is constructed and the breakeven point is identified. The break
even point is the point at which the cost is equal to the revenue. The return on investment
(ROI) is calculated as the following formula:
ROI = (Total Revenue - Total cost)
Table 10. Calculation of rn of Investment.
Year 2009 2010 20
Cost ($) 140,000 90,000 90,000 90,000 90,000
Revenue ($) 0 200,000 200,000 200,000 200,000
Loss/Profit ($) 0,000 -14 110,000 110,000 110,000 110,000
ROI -100% 120% 120% 120% 120%
28
If the company does not have other revenues besides the annual licensing fee,
then the revenue curve will be flat and fixed at $200,000 a year.
Breakeven analysis
050
100150200250
2,008 2,009 2,010 2,011 2,012 2,013 2,014
Year
Thou
sand
dol
lars
Total costRevenue
Figure 8. Breakeven Analysis
From the plot seen in Figure 8, we can determine that the company will make
profit by the end of the second year.
The Norden Rayleigh curve will help us to acess the financial flow, hence, control
the cost and budget. Figure 9 will show the NR curve predicting the funding profile
during the project’s first year, and Figure 10 will show the cumulative funding over time.
The calculation for the cumulative cost driver is shown in Table 11.
Table 11. Calculation of Normalized Accumulative Cost Driver
Technical risks Personal skills Competitive advantages Normalized cost driver
A=0.006 A=0.002 A=0.001 A=0.03
29
Funding Profile in the First Year
0
5000
10000
15000
20000
25000
0 5 10 15
Months
Dol
lars
Dollars
Figure 9. Funding Profile in the First Year
Cumulative Funding in the First Year
0
50000
100000
150000
0 5 10 15
Months
Dol
lars
Dollars
Figure 10. Accumulative Funding in the First Year.
30
4.13 Exit Strategy
Instead of licensing out our technology to other pharmaceutical manufacturing
companies, we might sell the patented technology, and work on other oral drug delivery
technologies. Alternatively, we might merge with our customers or competitors, which
want to complement their drug delivery technology portfolio or extend their drug patent
by our novel drug delivery technology
31
CHAPTER 5: PROJECT SCHEDULE
We divide our project into two phases: one-semester preparation phase and one-
semester experimental phase. The first phase involves concept development, literature
search, and project justification, allocation of budget, equipment assembly and validation.
The last phase involves experiment and data collection with presentation of results in
report form. The date and duration of each event is listed in Table 12 and 13.
Table 12. The Gantt chart of the resulting schedule is shown in Table 13.
Table 12. Project schedule
32
Table 13. The Gantt chart of the project schedule.
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33
CHAPTER 6: RESULTS and DISCUSSION
6.1 Assessment of Microcapsule Size
From the results of initial OFAT screening tests , the optimal range for FA was
determined to be - 2.6- 3.2 L/min at FL=0.3 ml/min.. Lower liquid and air flow rates
produced non-spherical beads with undesired tails. Higher values of FL and FA resulted in
non-uniform atomization and leaky beads. Higher values of Tliq resulted in disintegrated
microcapsules; hence the maximum liquefaction time was set to 4 minutes. Thus,
experiments were conducted according to the conditions summarized in Table 5 at a
constant liquid flow rate (FL=0.3 ml/min). The microcapsule size is measured prior to the
polycation adsorption step because the thickness of the layer (5-10 µm) is negligible
compared to the total microcapsule diameter (d≈500 µm).
Microcapsule sizes cluster by air flow rate as shown in Figure 11- 13. Figure 14-
16 represents the microcapsule size distributions for FA ranging from 2.6-3.2 L/min. For
all three frequency distributions the mean, median, and mode are the same indicating that
the data is normal. Using upper and lower specification limits (USL=700 µm and
LSL=300 µm) for a 500 ±200 µm specification, the percentage defective ranges from
100% to 0 % with two-sided process capabilities (Cp) close to 3.0, for FA ranging from
2.6-3.2 L/min. The process capability results suggest highly reproducible
microencapsulation conditions across the experimental matrix.
34
Figure 11. Microcapsule with mean size of 732 µm (FL-0.3 ml/min, FA – 2.6 L/min)
Figure 12. Microcapsule with mean size of 633 µm (FL-0.3 ml/min, FA – 2.9 L/min)
35
Figure 13. Microcapsule with mean size of 543 µm (FL-0.3 ml/min, FA – 3.2 L/min)
36
Figure 14 Frequency distribution of 60 microcapsules (FL- 0.3 ml/min and FA- 2.6 L/min)
37
Figure 15. Frequency distribution of 60 microcapsules (FL- 0.3 ml/min and FA- 2.9 L/min)
38
Figure 16. Frequency distribution of 60 microcapsules (FL- 0.3 ml/min and FA- 3.2 L/min) 6.2 Assessment of the effect of airflow, poly-cation concentration, adsorption time, and liquefaction time on the size and signal/noise ratio of the microcapsule
The two types of analysis performed are the average e and the signal to noise
(S/N) ratio conducted on the microcaspule size characteristic. The S/N ratio is a stastiscal
measure of performance used in evaluating the quality of the product. The signal to noise
ratio measures the level of performance and the effect of noise factors on the
performance. It is a objective measure of quality that takes both the mean and the
variance into account for the evaluation of the stability of a process. The three standard
types of signal to noise ratio are: a) smaller the better, b) bigger the better, and c) nominal
39
the better. In our experiment we calculated the signal to ratio for nominal the better type.
In the parameter design for nominal the best, the two things that need to be determined
are a) signal to noise ratio S/N (db) as a measure of the change in the variability and b)
sensitivity Sm (db) as a measure of change in the mean. The formulas to calculte the
sensitivity and S/N ratio are shown below:
Total sum of n variables =
Sensitivity = Sm (db) = 10 log Sm
= 10 log
The sensitivity and signal to noise ratio of 60 randomly selected microcapsules
sizes from 1-9 batches (Taguchi matrix) have been calculated calculated. The effects of
air flow rate (FA), polycation concentration[cation], adsorption time (Tads) of polycation,
and liquefaction time (Tliq) on the microcapsule size have been computed. Results have
been summarized in Table 14 and Figure 17-18.
40
Table 14. Summary of Taguchi Analysis of Microcapasule Size Characterustic using the Nominal the Best Optimization
Average FA [Cation] Tads Tliq
level1 728.0 635.4 635.4 635.4 level2 626.9 633.3 633.3 633.3 level3 547.0 633.3 633.3 633.3 Range 181.0 2.1 2.1 2.1
% 96.6 1.1 1.1 1.1 S/N FA [Cation] Tads Tliq
level1 30.5 28.5 28.5 28.5 level2 26.0 28.5 28.5 28.5 level3 28.9 28.5 28.5 28.5 Range 4.6 0.0 0.0 0.0
% 98.4 0.5 0.5 0.5
The effect of air flowrate leads the pareto of effects in the Average and S/N
analyses as presentd in Table 14. As shown in Figure 18, the S/N ratio for the
microcapsule size reaches a minimum at FA=2.9 L/min, however the ratios are
comparable at levels 1 and 3. Since operating at level 3 (FA=3.2 L/min) yields the target
size of 500 ±200 µm with 0% defect, , the recommended operating conditions for
microcapsule size is FA=3.2 L/min, at FL=0.3 ml/min independent of the level of other
factors.
41
Figure 17. Effect of air flow rate poly cation concentration, adsorption time, and liquefaction time on microcapsule size
42
Figure 18. Effect of air flow rate poly cation concentration, adsorption time, and liquefaction time on microcapsule signal/noise ratio 6.3 Assessment of leakage
Blue dextran (Mv= 2,000,000) leakage is a measure of the membrane strength.
The average molecular weight cutoff reported for APA microcapsules is 100,000; hence
blue dextran leakage results from a weak membrane. A Blue dextran calibration curve
was obtained for a concentration range of 0-1 mg/ml using a UV-VIS spectrophotometer
(HP8453) operating at λ=280 nm. The absorbance results were plotted as shown
in Figure 19. Microcapsules from all 9 Taguchi runs were stored in 5ml of saline for 48
hrs at 4 ºC. 1 ml of the supernatant was pipetted out and dispensed into a glass cuvette
for absorbance measurements at t=0, 24 and 48 hrs. The resultant absorbance data w
compared to the blue dextran calibration curve for determining the extent of leakage. The
as
43
results from our analysis were compared to 0.1 mg/ml of blue dextran. Absorbance
results for batches 1-9 were much lower than 0.1 mg/ml as shown in Figure 20. The
leakage is undetectable by this analytical method. Hence the microcapsules were very
robust and there was no leakage at least for 48 hours after the preparation.
Figure 19. Blue dextran calibration curve
44
Figure 20. Blue dextran leakage as a measure of microcapsule strength
6.4 Assessment of pH resistance
The pH susceptibility of the microcapsules was tested by incubating the
microcapsules in simulated Gastric fluid (SGF) and Simulated Intestinal fluid (SIF). In
addition to poor pH resistance for APA microcapsules, screening experiments showed
that the ideal size for testing membrane pH resistance was approximately 1000 µm for
thorough detection of membrane damage given our experimental capabilities. Due to
these restrictions Alginate-Chitosan-Alginate (ACA) microcapsules were prepared
following a recommended combination of variables outside of the L9 matrix (Mobed-
Miremadi et.al, 2009). 200µl of ACA and APA microcapsules were incubated at 37 ºC
and agitation at a rate of 150 rpm in 10 ml of SGF solution for 2 hrs. After 2 hrs, the
microcapsules were extracted from the SGF solution and split into 2 batches. Batch 1
was examined for visual changes. Batch 2 was incubated in an SGF/SIF solution
45
(1ml/10ml) for 18 hrs in order to simulate the gastro-intestinal transit. Similarly, 200 µl
of ACA and APA microcapsules were incubated at 37 ºC and agitation rate of 150 rpm in
10 ml of SIF for 2 hrs.
The APA and ACA microcapsules remained intact after intact in the SGF fluid
however, the surface of the APA microcapsule appears wrinkled as seen in Figure 21and
22. The wrinkles are due to solubility decrease and not enzymatic degradation. Both
ACA and APA microcapsules remained intact in the SIF solution. After 18 hours of
incubation in SIF, the ACA microcapsule was found to be intact, while APA
microcapsule disintegrated as shown in Figure 23 and 24.
Figure 21. ACA microcapsules after 2 hr incubation in SGF
46
Figure 22 APA microcapsules after 2 hr incubation in SGF
Figure 23 Swollen ACA Microcapsules after 2 hrs Incubation in SGF followed by 18 hrs Incubation in SIF
47
Figure 24. Dissolved APA Microcapsules after 2 hrs Incubation in SGF followed by 18 hrs Incubation in SIF
6.5 Prediction Model
The main objective of the Taguchi design was to identify the operating parameters
and operating levels for reproducible robust capsule formation. The ideal models are
proposed below:
48
liqliq
adsads
A
liqliq
adsads
AA
liqliq
adsads
AA
TofleveltcoefficienTxaTofleveltcoefficienTxa
CationleveloftcoefficienCationxaFAofleveltcoefficienFxa
erceptawhere
xaxaxaxaaopHY
valueTtcoefficienTxavalueTtcoefficienTxa
valueCationtcoefficienCationxavalueFtcoefficienFxa
erceptawhere
xaxaxaxaaoStrengthY
valueTtcoefficienTxavalueTtcoefficienTxa
valueCationtcoefficienCationxavalueFtcoefficienFxa
erceptawhere
xaxaxaxaaosizeY
/:4,"4/:3,"3
][/][:2,"2/:1,"1
int:"0
4"43"32"21"1"
/:4,'4/:3,'3
]/[][:2,'2/:1,'1
int:0'
4'43'32'21'1'
/:4,4/:3,3
]/[][:2,2/:1,1
int:0
44332211
++++=
++++=
++++=
Since no change was detected in leakage and the pH susceptibility had to be taken
out as a response due to our experimental capabilities, the only response left is
microcapsule size. As discussed in section 6.2, FA accounts for over 95% of the effects.
Hence a first order linear model plotted in Figure 25 can be used to accurately (r2=0.995)
predict the size in terms of air flow rate.
181551.90 xsizeY +−=
49
Figure 25. First order linear model plot to predict the size of the microcapsule in terms of air flow rate.
50
CHAPTER 7: CONCLUSION
Among many potential applications of microencapsulation technology is the use
of microcapsules as oral drug delivery vehicle for treatment of metabolic inborn errors
such as phenylketonuria. Alginate- Polycation-Alginate microcapsules such as APA or
ACA microcapsules must be mechanically and chemically strong under the physiological
conditions. Air flowrate is the main factor controlling microcapsule size as proven by
results of the L9 Taguchi matrix. The recommended operating conditions for the target
microcapsule size of 500 ±200 µm are FA=3.2 L/min, at constant FL=0.3 ml/min
independent of the level of other experimenatalfactors. A first order linear model
correlating microcapsule size to air flow rate was sufficient to predict the microcapsule
size over the experimental range (FA=2,6-3.2 L/min ). The micrcapsules were
mechanically robust and there was no leakage detected at 4º C after 48 hours of
preparation.
In terms of susceptibility to pH , the ACA microcapsules survived the simulated
gastro-intestinal transit intact compared to disintegrated APA microcapsules. Hence
Chitosan is the recommended polycation for oral delivery of microcapsules.
With the human genome being mapped out completely, as more genetic diseases
such as IEMs are surfacing, the oral delivery approach using microencapsulation will
gain an even greater importance.
51
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Maryam Mobed-Miremadi, Arathi Asthi, Raki Nagendra, Varun Varma (2009).
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APENDIX A
1. 1.5% Sodium Alginate Solution Dissolve 3g of A2033 Alginate in 200 ml of 0.9% NaCl. Dissolve Overnight
2. 0.9% Nacl
Dissolve 9g of NaCl in 1000 ml of DI water
3. 1.5% Cacl2 Dissolve 15g of Cacl2 in 1000 ml of DI water
4. 0.0.25% PLL
Dissolve 50mg of PLL in 200ml NaCl (0.9%)
5. 1.5% Sodium Citrate Dissolve 7.5g in 500ml of DI water
6. Blue Dextran Solution Dissolve 25mg of Blue dextran per ml of 0.9% NaCl
7. Blue dextran and Alginate Dissolve Blue dextran and alginate in the ratio of 30:70 respectively
8. 0.1% Nag (4 min) Dissolve 0.4g of A0682 (low viscosity) alginate in 400ml of 0.9% NaCl.