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

iii

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.

iv

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

v

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 

vi

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 

vii

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 

viii

ix

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

1

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

2

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.

3

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

4

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.

5

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

6

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

7

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

8

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).

10

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)

11

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

12

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.

13

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.

Buy SmartDraw!- purchased copies print this document without a watermark .

Visit www.smartdraw.com or call 1-800-768-3729.

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

REFERENCES

BioMarin Pharmaceutical Inc. (2008, February 28). Form 10-K. Retrieved November

2009 from Morningstar Document Research database

Bressel T.A.B., Paz A.H., Baldo G., et al. (2008). An effective device for generating

alginate microcapsules. Genetic Molecular Biology, 31(1), 136-140.

Gugerli R.E., Cantana C., Heinzen U., et al. (2002). Quantitative study of the production

and properties of alginate/poly-L-lysine microcapsules. Journal of

microencapsulation, 19(5), 571-590.

Kamyshny, A., & Magdassi S. (2006). Microencapsulation. In P. Somasundaran (Eds.),

Encyclopedia of surface and colloid science (Vol. 5, pp. 3957-3970). CRC Press.

Klein H. E., Sundstrom D. W., & Shim D. (1985). Immobilization of enzymes by

microencapsulation. Immobilized Cells and Enzymes. A Practical Approach, J.

Woodward (pp.85). IRL Press.

Lin J., Yu W., Liu X., et al. (2008). Vitro and in vivo characterization of alginate-

chitosan-alginate artificial microcapsules for therapeutic oral delivery of live

bacterial cells. Journal of bioscience and bioengineering, 105(6), 660-665.

Lindner M. (2007). BioMarin surges on FDA okay. Retrieved on April 26, 2009 from

http://www.forbes.com/2007/12/14/biomarin-pharmaceuticals-closer-markets-

equity-cx_ml_1214markets32.html

Matricardi P. (2008). Recent advances and perspectives on coated alginate microspheres

for modified drug delivery. Journal: Expert opinion on drug delivery, 5(4), 417-

425.

52

Maryam Mobed-Miremadi, Arathi Asthi, Raki Nagendra, Varun Varma (2009).

Alginate-chitosan-alginate microcapsules for oral administration. 2009 AIChE

Annual Meeting, Nashville, TN

Maryam Mobed-Miremadi and Craig Stauffer (2009). Bio-printing and permeability of

artificial cells. IMD conference, San Jose, California

Melvik J.E., & Dornish M. (2004). Alginate as a carrier for cell immobilization. In V.

Nedovic & R. Willaert (Eds), Fundamentals of Cell Immobilisation Biotecnology

(pp. 33-53). Kluwer Academic.

OralSolve ® (n.d). Retrieved November 15, 2009, from website:

http://www.cimalabs.com/technology/orasolv

Phillips C.R., & Poon Y.C. (1988). Methods of cell immobilization. Immobilization of

cells (pp.11-75). Springer-Verlag.

Ray S. (2007). Long road to developing a PKU therapeutic. Retrieved on April 20, 2009

from www.pkunews.org/research/LongRoad.htm

Riddle K.W., & Mooney D.J. (2004). Biomaterials for cell immobilization. In V. Nedovic

& R. Willaert (Eds), Fundamentals of Cell Immobilizations Biotechnology (pp.

22-25). Kluwer Academic.

Socaciu C. (2007). Updated technologies for extracting and formulating food. In C.

Socaciu (Ed), Food colorants: chemical and functional properties (pp. 303-329).

CRC Press.

Technology & Products (n.d)) Retrieved November 15, 2009, from website:

http://www.particledynamics.com/products/default.asp

53

Technology: Microcaps ®. (n.d). Retrieved November 15, 2009, from website:

http://www.eurand.com/tech_microcaps.html

Thu B., Bruheim P., Espevik T., et al. (1996). Alginate polycation microcapsules,

Biomaterials, 17(10), 1031-1040.

Torrado J.J., & Augburger L.L. (2008). Tableting of multiparticulate modified release

systems. In L.L. Augsburger & S.W. Hoag (Eds), Pharmaceutical Dosage

Forms: Tablets (Vol. 2: Rational Design and Formulation, pp. 509-511). Informa

Health Care.

Visakorpi J. K., Palo J., & Renkonen O.V. (2008). The incidence of PKU in Finland.

Acta Pædiatrica, 60(6), 666 – 668. 

Vos V., Bucko M., Gemeiner P., et al. (2008). Multiscale requirements for

bioencapsulation in medicine and biotechnology. Biomaterials, 30(13), 2559-

2570.

Yoon Y., Baek N., & Park K. (2001). Microencapsulation methods for delivery of protein

drugs. Biotechnology and Bioprocess Engineering, 6(4), 213-230.

54

55

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.