Pullulan ω-carboxyalkanoates for Drug Nanodispersions ...

Pullulan ω-carboxyalkanoates for Drug Nanodispersions

Jameison Theophilus Rolle

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Master of Science

In

Macromolecular Science and Engineering

Kevin J. Edgar, Committee Chair

Richey Davis

Lynne S. Taylor

July 30th, 2015

Blacksburg, VA

Keywords: pullulan, amorphous solid dispersions, carboxyalkanoates, drug delivery

Copyright 2015, Jameison T. Rolle

Pullulan ω-carboxyalkanoates for Drug Nanodispersions

Jameison T. Rolle

Abstract

Pullulan is an exopolysaccharide secreted extracellularly by the black yeast-like fungi

Aureobasidium pullulans. Due to an α-(1→6) linked maltotriose repeat unit, which interferes with

hydrogen bonding and crystallization, pullulan is completely water soluble unlike cellulose. It has

also been tested and shown to possess non-toxic, biodegradable, non-mutagenic and non-

carcinogenic properties. Chemical modification of polysaccharides to increased hydrophobicity

and increase functionality has shown great promise in drug delivery systems. Particularly in

amorphous solid dispersion (ASD) formulations, hydrophobicity increases miscibility with

hydrophobic, crystalline drugs and carboxy functionality provides stabilization with drug moieties

and well as pH specific release. Successful synthesis of cellulose ω-carboxyalkanoates have been

reported and showed great promise as ASD polymers based on their ability to retard the

recrystallization of the HIV drug ritonavir and antibacterial clarithromycin. However, these

cellulose derivatives have limitations due to their limited water solubility. Natural pullulan is

water-soluble and modification with ω-carboxyalkanoate groups would provide a unique set of

derivatives with increased solubility therefore stronger polymer-drug interactions in solution.

We have successfully prepared novel pullulan ω-carboxyalkanoates, which exhibit good

solubility in polar aprotic and polar protic solvents. All derivatives exhibit high thermal stability

and most recorded high glass transition temperatures. Due to unknown impact of their three

dimensional structure on miscibility and stabilization of drug against crystallization, each of these

polymers possesses great potential for use in various drug delivery applications.

iii

Dedication

This thesis is dedicated to my parents and family members who were always so supportive of my

decisions and always encouraged me to continue to do my best.

iv

Acknowledgements

I would first like to give thanks and praise to God, because without Him none of this

would have been possible and it is because of Him, through stressful and doubtful times that I

was able to get through.

I would also like to thank my advisor Dr. Kevin Edgar for allowing me the opportunity to

work with his group during my time here at Virginia Tech. He has always believed in me and

always made me feel A LOT less stressed after our research update meetings. I am truly thankful

for all the advice he has given over these past 2 ½ years and although I am not pursuing a PhD, I

am grateful for him being so supportive with my decision to attend medical school. I will always

cherish the moments that I have been blessed to work with him and I am thankful for all I have

learned.

I would also like to thank Dr. Richey Davis and Dr. Lynne Taylor for serving as members

of my committee and for their support and guidance.

I would like to that Dr. Judy Riffle and all of my classmates in the Fall 2013 MACR class.

They made the transition from undergraduate to graduate life so smooth, I will cherish all of

these friendships, and the memories made studying for those MACR exams.

I would also like to thank all the members of the Edgar Research Group. We have had so

much fun during my time here and I am deeply appreciative of the support of each of them. I am

so happy to have been able to become such close friends and I hope that they all continue to

strive for excellence. I thank them for all their guidance and direction and wish them the very

best.

Finally, I would like to that the Institute for Critical Technology and Applied Science

(ICTAS), Macromolecules and Interfaces Institute (MII), the Department of Sustainable

Biomaterials and the National Science Foundation (NSF) for all of their support during my time

at Virginia Tech.

Thank You Everyone!!!

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Table of Contents

Abstract ................................................................................................................................ ii

Dedication ............................................................................................................................ iii

Acknowledgements .............................................................................................................. iv

List of Figures ...................................................................................................................... vii

List of Tables ........................................................................................................................ ix

CHAPTER 1: Introduction ....................................................................................................... 1

CHAPTER 2: Review of Literature ........................................................................................... 4

2.1 History of Pullulan ......................................................................................................................... 4

2.2 Production of Pullulan .................................................................................................................. 5

2.3 Properties and Applications of Pullulan ........................................................................................ 9

2.4 Chemical Modification ................................................................................................................ 11

2.5 Drug Delivery............................................................................................................................... 22

2.6 Conclusion ................................................................................................................................... 26

2.7 References .................................................................................................................................. 27

CHAPTER 3: Synthesis of Pullulan ω-carboxyalkanoates for Drug Nanodispersions ............... 38

3.1 Abstract ....................................................................................................................................... 38

3.2 Introduction ................................................................................................................................ 38

3.3 Experimental ............................................................................................................................... 42

3.3.1 Materials ............................................................................................................................ 43

3.3.2 Measurements ................................................................................................................... 43

3.3.3 Synthesis ............................................................................................................................. 45

3.4 Results and Discussion ................................................................................................................ 50

3.4.1 Synthesis of pullulan ω-carboxyalkanoates ............................................................................... 50

3.4.2 Thermal properties .................................................................................................................... 57

3.4.3 Solubility ..................................................................................................................................... 60

3.4.4 Solubility parameters ................................................................................................................. 61

3.5. Conclusion ................................................................................................................................... 62

3.6 Acknowledgements ..................................................................................................................... 63

3.7 References .................................................................................................................................. 63

CHAPTER 4: Summary and Future Works ............................................................................. 67

4.1 Summary ..................................................................................................................................... 67

vi

4.2 Future Works .............................................................................................................................. 68

4.2.1 Synthesis of pullulan ω-carboxyalkanoates by olefin cross-metathesis (OCM) ................. 68

4.2.2 Amorphous Solid Dispersions ............................................................................................. 69

4.2.3 Other Uses........................................................................................................................... 71

4.3 References .................................................................................................................................. 72

APPENDIX............................................................................................................................ 73

vii

List of Figures

Figure 2.1: Chemical structure of pullulan showing the maltotriose repeat unit as well as the

trisaccharide repeat unit …………………………………………………………………………………………………………. 5

Figure 2.2: Proposed biosynthetic pathway for pullulan production utilizing glucose as well as

other polysaccharides as a food source. Adapted from Chen et. al. ……………………………………... 6-7

Figure 2.3: Chemical structure of carboxymethyl pullulan substituted at various positions ..…. 13

Figure 2.4: Depiction of cholesterol modified pullulan (CHP) and the structural differences of

the labile (acL-CHP) and stable (acS-CHP) derivatives. Differences in structure are shown in red.

Adapted from Morimoto et.al. ………………………………………………………………………………………………. 16

Figure 2.5: Schematic of chloroformate activation to yield carbamate-containing derivatives.

Adapted from Bruneel …………………………………………………………………………………………………………… 18

Figure 2.6: Synthesis of pullulan monosuccinate (SUPA) and the prodrug CP-SUPA via

coordination, adapted from Wang et. al. ……………………………………………………………………………… 19

Figure 2.7: Regioselective synthesis of 6-amino and 6-amido pullulan esters ……………………….. 20

Figure 2.8: Oxidation of primary hydroxyl groups using the 4-acetamide TEMPO/NaClO/NaClO2

system adapted from Tamura et. al. ……………………………………………………………………………………… 21

Figure 2.9: Synthesis of 6-carboxypullulan ethers via TEMPO oxidation ………………………………… 22

Figure 3.1: Scheme of the synthesis of pullulan ω-carboxyalkanoates …………………………………… 42

Figure 3.2: (a) 1H NMR and (b) 13C NMR of monobenzyl pullulan propionate suberate (DSω-carboxy

= 0.68) in d6-DMSO and CDCl3 respectively. …………………………………………………………………………… 53

Figure 3.3: ATR-FTIR spectra of monobenzyl pullulan suberate propionate …………………………….54

Figure 3.4: (a) 1H NMR and (b) 13C NMR of monobenzyl pullulan propionate suberate (DSω-carboxy

= 0.68) in d6-DMSO showing the absence of phenyl and benzyl peaks. 10* denotes the carbonyl

carbon adjacent to the ω-carboxy group. ……………………………………………………………………………… 55

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Figure 3.5: Comparison of the ATR-FTIR spectra of pullulan suberate

acetate/propionate/sebacate and their stretches relative to each other ………………………………. 56

Figure 3.6: DSC thermograms of pullulan suberate derivatives showing the Tg as well as

PullPrSeb0.99 showing endotherm. ………………………………………………..…………………………………….. 59

Figure 3.7: TGA thermograms of pullulan suberate derivatives ……………………………………………… 60

Figure 4.1: Schematics of synthesis of cellulose ω-carboxyalkanoates via olefin cross-metathesis

adapted from Meng et. al. …………………………………………………………………………………………………….. 68

Figure 4.2: Schematic of how an amorphous solid dispersion works ……………………………………… 70

ix

List of Tables

Table 2.1: Comparison of the variation of pullulan obtained from different microorganisms ..… 8

Table 2.2: Solubility of pullulan in various solvents ………………………………………………………………… 10

Table 2.3: Chemical modification of pullulan and their potential/currents use ……………………… 12

Table 2.4: Comparison of the solubility of esterified and etherified pullulan with similar DS .... 15

Table 3.1: Comparison of DS ω-carboxy, DS alkanoate, and hydroxyl group content of each pullulan

derivative ………………………………………………………………………………………………….…………………………… 51

Table 3.2: TGA and DSC values for all pullulan ω-carboxyalkanoates and their solubility

parameters …………………………………………………………………………………….…………………………………….. 58

Table 3.3: Solubility of pullulan ω-carboxyalkanoates in various solvents ……………………………… 61

1

CHAPTER 1: Introduction

Pullulan is a naturally synthesized polysaccharide secreted extracellularly by the black

yeast-like fungi Aureobasidium pullulans1. As an exopolysaccharide, the main function of pullulan

is to act as protection against desiccation and attack from other organisms that may prey on the

fungi by forming a biofilm2. Additionally, it also functions to regulate the diffusion of gases and

molecules into and out of the cellular environment. In commercial use, the main applications of

pullulan take advantage of its film-forming capabilities. The films are edible, clear, tasteless and

odorless and are used in a large amount of food applications such as candies, confections and

Listerine Pocket Packs3. In the pharmaceutical industry, pullulan is of great interest due to its

non-toxicity, biodegradability, biocompatibility and non-carcinogenic properties4-5.

Water solubility is one of the many properties that pullulan has that differentiates it from

other more well-known polysaccharides6. However, most chemical modifications aim to decrease

its water solubility in order to increase its range of uses for biomedical applications7. The main

goal of this thesis is to synthesize a range of pullulan ester derivatives with varying

hydrophobicities to be used in drug delivery systems. These derivatives will contain enhanced

functionality by introducing pH sensitivity, carboxylic acid groups, to the pullulan backbone,

which will afford a release mechanism that can be utilized by various modes of drug delivery,

including oral and intravenous avenues.

In the literature, successful synthesis of cellulose ω-carboxyalkanoates has already been

reported and these polymers have shown extremely promising results in amorphous solid

dispersion (ASD) formulations8-10. Increased hydrophobicity imparts miscibility with hydrophobic,

crystalline drugs, and carboxyl functionality provides specific interactions with drug moieties, as

well as pH-controlled release in the small intestine. Additionally, these cellulose derivatives have

been reported to show retardation of crystallization of many drugs, including the anti-HIV drug

ritonavir and the antibacterial clarithromycin. These properties, along with low toxicity and high

glass transition temperatures (Tg), make these cellulose -carboxyalkanoates promising ASD

polymers, but with limitations due to their limited water solubility. Native pullulan however, is

2

water soluble due to an α-(1→6) linked maltotriose repeat unit, whose -stereochemistry and

mixed (1→4), (1→6) linkages interfere with packing, hydrogen bonding, and crystallization.

Therefore, pullulan is a promising template for similar modification, which we hypothesize would

give rise to a set of derivatives with enhanced solubility, permitting stronger polymer-drug

solution interactions. Even beyond the use as an ASD polymer for oral drug delivery, these

derivatives show great promise for intravenous formulations as well, given the ability of pullulan

to be cleared from circulation, which cellulosic polymers lack.

An outline for this thesis is as follows: Chapter 2 will discuss the background and history

of pullulan and its many uses in the food and pharmaceutical industries. Additionally, a detailed

outline of the chemical modifications of pullulan throughout the literature is presented, and

some of the various applications for which pullulan and its derivatives have been adapted are

described. The end of the chapter will talk in detail about the various modes of drug delivery as

well as their limitations and the promise of ASD formulations in oral administration. Chapter 3

will go into the detailed synthesis of pullulan ω-carboxyalkanoates as well as their properties and

the promise they have for incorporation into drug delivery systems, specifically in their roles as

ASD polymers. Chapter 4 will provide the summary of the research as well as a suggested course

for the implementation of these derivatives for ASDs as well as for nanoaggregates in other

delivery formulations.

REFERENCES

1. Leathers, T. D., Biotechnological production and applications of pullulan. Appl. Microbiol. Biotechnol. 2003, 62 (5-6), 468-473.

2. Bender, H.; Lehmann, J.; Wallenfels, K., Pullulan, an extracellular glucan from Pullularia pullulans English summ. Biochim Et Biophys Acta 1959, 36 ((2)), 309-316.

3. Shih, F. F., Edible films from rice protein concentrate and pullulan. Cereal Chem. 1996, 73 (3), 406-409.

4. Kimoto, T.; Shibuya, T.; Shiobara, S., Safety studies of a novel starch, pullulan: Chronic toxicity in rats and bacterial mutagenicity. Food and Chemical Toxicology 1997, 35 (3-4), 323-329.

3

5. Fujii, B.; Shinohara, S., Polysaccharide produced by aureobasidium-pullulans ferm-p4257 ii. Toxicity test and antitumor effect. Bulletin of the Faculty of Agriculture Miyazaki University 1986, 33 (2), 243-248.

6. Singh, R. S.; Saini, G. K.; Kennedy, J. F., Pullulan: Microbial sources, production and applications. Carbohydr. Polym. 2008, 73 (4), 515-531.

7. Cheng, K.-C.; Demirci, A.; Catchmark, J. M., Pullulan: biosynthesis, production, and applications. Appl. Microbiol. Biotechnol. 2011, 92 (1), 29-44.

8. Liu, H. Y.; Cherniawski, B. P.; Kar, N.; Edgar, K. J., Synthesis of carboxyl-containing long chain cellulose esters. Abstr. Pap. Am. Chem. Soc. 2012, 243, 1.

9. Liu, H. Y.; Ileybare, G. A.; Cherniawski, B. P.; Ritchie, E. T.; Taylor, L. S.; Edgar, K. J., Synthesis and structure-property evaluation of cellulose omega-carboxyesters for amorphous solid dispersions. Carbohydr. Polym. 2014, 100, 116-125.

10. Liu, H. Y.; Kar, N.; Edgar, K. J., Direct synthesis of cellulose adipate derivatives using adipic anhydride. Cellulose 2012, 19 (4), 1279-1293.

4

CHAPTER 2: Review of Literature

2.1 History of Pullulan

Pullulan is a natural polysaccharide, which is secreted extracellularly by the fungus

Aureobasidium pullulans (A. pullulans)1. It was first discovered by Bauer11 in 1938, but isolation

and characterization were not achieved until much later in 1958 by Bernier12. The name pullulan

was given by Bender in 1959 who determined, based on its positive optical rotation and infrared

spectrum that it was composed completely of α-D-glucans with predominantly α-(14)

linkages2. It was not until the 1960’s that the basic structure of pullulan was resolved13. Due to

the discovery of the enzyme pullulanase, which has specificity to hydrolyze the α-(16) linkages,

they discovered that the polymer was converted almost quantitatively to maltotriose units. Upon

further investigation and confirmation with infrared spectroscopy, periodate oxidation and

methylation data, it was determined that pullulan contained α-(14) and α-(16) linkages in

the ratio of 2:114. Therefore, pullulan is depicted as a linear polysaccharide having a maltotriosoyl

[α-D-Glcp-(14)-α-D-Glcp-(14) )-α-D-Glcp-(14)] repeat unit connected by α-(16) linkages.

The trisaccharide repeat unit is represented by the nomenclature [4)-α-D-Glcp-(16)-α-D-

Glcp-(14)-α-D-Glcp-(1] (Figure 2.1).

Pullulan however, can also be described as a polymer of panose [α-D-Glcp-(16)-α-D-

Glcp-(14)-α-D-Glcp] and isopanose [α-D-Glcp-(14)-α-D-Glcp-(16)- α-D-Glcp] subunits,

which may be more accurate with respect to the biosynthesis of the polymer7, 15. Moreover, it

was found that pullulan contains a small percentage of maltotetrose subunits [α-(14)-Glcp-α-

(14)- Glcp-α-(14)-Glcp-α-(16)-Glcp] by Catley16 and coworkers. The structure of pullulan is

slightly affected by this minor percentage of maltotetrose groups, but the effect of this small

percentage and the overall physio-chemical properties of the polysaccharide may be minimal.

These variations in structure are however the reason why the polymaltotriose repeat unit

produced by Aureobasidium pullulans and the other variations produced by other microbes

which include maltotetrose units are all referred to as “Pullulan” in the literature.

5

Figure 2.1: Chemical structure of pullulan showing the maltotriose repeat unit as well as the trisaccharide repeat unit

2.2 Production of Pullulan

Pullulan is primarily produced from the fungus Aureobasidium pullulans (formerly

Pullularia pullulans). Found usually as an epiphyte or endophyte in the environment, it usually

resides in damp places such as soil, water, forests, decaying litter, wood and other plant

materials6. The fungus does contain degradative enzymes such as amylases, proteases, lipases,

esterases and hemicellulases17. Since the A. pullulans is a polymorphic fungus, it has three

distinctive forms. They include elongated branched septate filaments, large chlamydospores and

smaller elliptical yeast-like cells. Due to the production of a dark, melanin-like compound, the

fungus appears dark green to black in color. A drawback during pullulan production is that

molecular weight is lost as the fermentation progresses during submerged growth18. There has

been a lot of interest in relating pullulan production based on the morphological form of the

6

fungus used. Various studies have been conducted and have shown that blastospores, which are

a consequence of the shift from mycelial to unicellular morphology, are responsible for the

production of pullulan6. However, research has also shown that when significant amounts of

chlamydospores are present, pullulan production proceeds regardless of culture conditions.

Which morphological form is best in pullulan production continues to be a point of debate

throughout the literature19-20.

There has been extensive investigation in the literature on the biosynthetic mechanism

of pullulan production of A. pullulans, but it is not yet fully understood6-7. It has been reported

that pullulan is synthesized intracellularly at the cell wall membrane and secreted to the cell

surface to form a slimy, loose layer20. One mechanism reported that the presence of three key

enzymes; α-phosphoglucose mutase, uridine diphosphoglucose pyrophosphorylase (UGDP-

pyrophosphorylase) and glucosyltransferase are important in pullulan production21. Catley and

McDowell22 attribute UDPG, a pullulan precursor, as an important nucleotide for pullulan

production as it initiates the attachment of a D-glucose residue to a lipid hydroperoxide via a

phosphoester linkage. Further transfer of the D-glucose from UDGP gives rise to an isomaltose

unit linked to the lipid. Finally, the isomaltose reacts with a lipid-linked glucose to form isopanose,

which is polymerized to form the pullulan chain Figure 2.2. It has been reported in the literature

that other sugars could be used as a carbon source for pullulan production as well, but the

formation pathways are not clear14, 23-24.

Monosaccharides Polysaccharides

Amylases

Fructose

2x ATP

Fructose 1,6

bisphosphate

Fructose 6

phosphate

Phosphorylation

Glucose 6

phosphate Isomerase

Glucose

Phosphorylation

n

Isomerases

α-phosphoglucose mutase

7

Figure 2.2: Proposed biosynthetic pathway for pullulan production utilizing glucose as well as other polysaccharides as a food source. Adapted from Cheng et. al.7

Not all strains of A. pullulans are able to produce pullulan and not all the pullulans

produced by these strains are structurally the same6. It has been reported that other forms of

microbes can also produce pullulan as well. Some of these microorganisms reported in the

literature to produce variations of pullulan include Tremella mesenterica, Cytaria harioti, Cytaria

darwinii, Cryphonectria parasitica, Teloschistes flavicans and Rhodototula bacarum6. The

Glucose 1

phosphate

UDPG-pyrophosphorylase

UDP-Glc

LPh UDP

LPh-Glc

LPh-Glc-(6←1)-Glc

UDPG

UDP

LPh-Glc-(6←1)-Glc-(4←1)-Glc

Isomaltosyl

Isopanosyl

Pullulan

glucosyltransferase

8

pullulans produced from these microbes do not entirely resemble pullulan formed from A.

pullulans, as they often contain different ratios of the glycosidic bonds, varying amounts of the

maltotetrose units, and small percentages of α-(13) units (Table 2.1).

Microbes Used Ratio of

α-(14): α-(16) linkages

Variations of Structure References

Aureobasidium pullulans*

2; 1.5 Contains up to 7% maltotetrose units; up to 6%

of α-(13) linkages

11, 16

Tremella mesenterica 2 25

Cytaria harioti 2.4 3-7 % α-(13) linkages 26

Cytaria darwinii 2 27

Cryphonectria parasitica 2.3-2.4 Up to 89% maltotetrose units

28

Teloschistes flavicans 1 29

*Different strains of A. pullulans produce pullulan with varying structural forms.

Table 2.1: Comparison of the variation of pullulan obtained from different microorganisms

Commercial pullulan production is mainly by the Hayashibara Company, which estimates

that approximately 300 metric tons of pullulan is produced annually7. Production starts with the

batch-wise cultivation of A. pullulans on a medium containing starch hydrolysates from dextrose,

usually 40-50 equivalents, at a concentration of 10-15%. Peptone, phosphate and basal salts

make up the medium used. The cultures are stirred and aerated at a constant temperature of 30

oC. The initial pH of the culture is adjusted to 6.5 which decreases to a final pH of 3.5 primarily

during the first 24 h. Within 75 h, maximum growth of the culture is obtained and by 100 h

optimal yield for pullulan is reached. The A. pullulans cells are removed via filtration of the dilute

culture broth. To ensure a product of high molecular weight and relatively free of melanin,

culture conditions as well as strain selection are very important. To remove the melanin, the

9

solution is treated with activated charcoal, and pullulan recovery and purification are achieved

through precipitation in primarily alcohols, although some other organic solvents can be used.

Other techniques such as ultrafiltration and ion exchange resins can be used for further pullulan

purification. The two types of pullulan sold commercially are food grade (US $20/kg) and

pharmaceutical grade (US $25/kg), which is just a deionized version.

There remain issues with the commercial production of pullulan. Due to the large amount

of melanin in Aureobasidium, removal is a problem and can lead to higher costs in production. In

addition, due to the large amounts of degradation enzymes present in the fungi, cultures in their

later stages suffer from decrease in pullulan molecular weight30. There have been many

published papers in regards to improving production cost of pullulan by means of less expensive

feedstock, isolation of improved production strains, or even developing alternative fermentation

schemes31-32. Studies have shown that sugars such as fructose, glucose, maltose, starch and

maltooligosaccharides support pullulan growth in A. pullulans. Similarly, carbon sources such as

beet molasses, cornmeal hydrolysates, corn syrup and grape skin pulp can also be used to

efficiently produce pullulan. A strain of A. pullulans, CFR-77, made from mixed-culture techniques

was used for production of pullulan using unrefined sugar from sugarcane juice33. It was reported

that the pullulan was pigment free and more viscous as compared with use of other sugars. Many

of these new technologies are geared towards a decrease in production costs, but until these

techniques are fully examined, pullulan has to be produced on a limited scale.

2.3 Properties and Applications of Pullulan

Dry pullulan is a white, non-hygroscopic powder that readily dissolved in hot or cold

water. Pullulan’s water solubility and applications are due primarily to the presence of the α-

(16) linkage which imparts flexibility to the polymer chains1. This also explains the tendency of

pullulan to form expandable flexible coils when in solution34. Due to this unique helix formation

ability, pullulan can exhibit a wide range of functions not normally seen in other polysaccharides.

Pullulan does exhibit solubility in DMSO, but it is poorly soluble in other organic solvents (Table

10

2.2). Other properties that make pullulan a keen area of research are its non-toxicity,

biodegradability, biocompatibility and non-mutagenic features4-5.

SOLVENT

Water DMSO DMF Pyridine Acetone Ethyl Acetate

THF Chloroform Toluene

Pullulan O O Δ Δ X X X X X

O Soluble; Δ Partially Soluble; X Insoluble

Table 2.2: Solubility of pullulan in various solvents35

In Japan, pullulan has been labeled as a generally recognized as safe (GRAS) food product.

It is odorless, tasteless and edible. In solution, its relatively low viscosity makes it suitable as a

filler and thickener for beverages and sauces as well as lotions and shampoos1. The viscosity,

however, is a function of molecular weight. Generally, pullulan has a number average molecular

weight (Mn) 100-200 kDa and weight average molecular weight (Mw) of 362-480 kDa which are

much lower than those of other polysaccharides. The reason for this lower molecular weight can

be attributed either to the differences in its biosynthetic pathway or the cell morphological

mechanism. The viscous solutions do not form gels and are stable over a broad pH range. When

dried, pullulan also has great adhesive abilities as well as foam retention properties when

dissolved in water36. It has been used as denture adhesives, food stabilizers and binders. Most

applications of pullulan in the food industry derive from its ability as a film former3. The pure

pullulan films readily dissolve and are used in the coatings of noodles, candies and confections.

Pullulan films are clear, highly oxygen-impermeable and have excellent mechanical properties,

which allow it to be of great use in packaging applications by preventing the oxidation of fatty

acids and vitamins in foods37. Pullulan can also be applied directly to foods as a glaze.

Pullulan can be used in place of starch to impart consistency, dispersibility and moisture

retention to foods38. It is superior to starch in retaining water, which allows for a longer shelf life

for products. Additionally partial replacement of starch with pullulan in foods like pastas and

11

baked goods also increases their shelf life due to the inability of bacteria, mold and fungi to

readily use pullulan as a carbon source, thereby reducing the rate of spoilage39. In humans,

pullulan is seen as dietary fiber and studies indicated that it functions as a prebiotic, which

promotes growth of beneficial bifidobacteria.

Pharmaceutical use of pullulan has been extensively studied40-42. The high concentration

of hydroxyl groups and the kinks in the chain due to the -linkages and the 1,6-linkages make

pullulan water-soluble, convenient for pharmaceutical applications. Capitalizing on its non-

toxicity, pullulan has been also used to form conjugates with vaccines, proteins and interferons.

This is also possible because of the ability of pullulan to be cleared from the body while not

invoking an immune response4. Due to its non-animal origin as well as being a natural product,

pullulan capsules comply with a variety of cultural and dietary group requirements, such as those

of vegetarians, diabetics, and patients with restricted diets. Pullulan does have a tendency to

accumulate in the liver, so many applications have been for liver-targeted drug and gene delivery

using pullulan moieties40. However, not all types of pullulan can be used in these applications.

Rapid increase in venous pressure when using pullulan above a molecular weight of 150 kDa was

observed7. Therefore, studies have shown that only pullulan with a polydispersity of 1.2 and

molecular weight of ≈ 60 kDa should be used for intravenous applications.

Other applications of pullulan have been in the areas of environmental remediation, due

to its ability to remove heavy metal ions from aqueous solutions; chromatography, as gel beads

as well as chromatography standards, and for the preservation of bacteria, through

immobilization and storage under certain conditions7.

2.4 Chemical Modification

The use of chemically modified pullulan has been widely reported throughout the

literature and is an important area of research (Table 2.3). Pullulan derivatives have been

successfully used as blood plasma substitutes, in drug and gene delivery, as antibacterial wound

12

dressings, and in cell wall biogenesis and cell proliferation and cluster formation7. Most

modifications aim to utilize its non-toxic, non-mutagenic, non-carcinogenic and biodegradable

properties. Due to its high water solubility, modifications are generally geared towards reducing

its hydrophilicity as well as imparting functionality to be of use in various applications. According

to the literature, pullulan has been esterified43, etherified44, sulfonated45, grafted46,

chlorinated47, blended48 and silylated49. Due to the large amount of free hydroxyl groups that

pullulan possesses, a large number of substituents can be attached. However, alcohol groups

tend to be not very reactive when exposed to certain chemical moieties, so modifications to

increase reactivity of either the pullulan backbone or the reagent itself are sometimes carried

out50.

Pullulan Derivative Potential/Current Use References

Pullulan alkyl esters Nanofibers 51

Vitamin B-6 bearing pullulan Protein nano-regulation 52

Pullulan acetate phthalate Microcapsules 53

Pullulan-spermine/DNA

anioplexes

Neuronal gene delivery 54

Carboxymethyl pullulan Thermoassociative particles

Hydrogels

55-56

Pullulan Sulfates Transmucosal protein delivery

Anticoagulants

57-58

Phosphorylated pullulan Implant surfaces 59

Succinylated pullulan

copolymers

Thermosensitive electrostatic complexes 60-61

Pullulan acetate Nanoparticles 62-63

Cholesteryl pullulan pH sensitive gels 64

Table 2.3: Chemical modifications of pullulan and derivatives’ potential/current use

13

Anionic modification of pullulan has been widely reported to be successful to create

targeting moieties for biomedical applications65-67. One of the most studied anionic pullulan

derivatives is carboxymethyl pullulan (CMP) (Figure 2.3). Successful synthesis of CMP has been

reported by reaction of sodium chloroacetate with pullulan in isopropyl alcohol68. Though the

reaction is not regioselective, substitution along the pullulan backbone was found to be more

favorable for the C-2 OH group. The reactivity order of the hydroxyl groups was determined by

1H NMR spectroscopy and found to be C-2 OH> C-4 OH> C-6 OH> C-3 OH. Thermoassociative

nanoparticles have also been made using Jeffamines attached to a periodate oxidized CMP67. The

amphiphilic nature of these derivatives afforded them the ability to retain hydrophobic,

hydrophilic and amphiphilic dyes. CMP-DOX nanoparticle conjugates have shown high pH

sensitivity and been used to target mouse fibroblast cells, human liver cancer cells as well as

human cervical carcinoma cells69. The hydrazone bond formed with hydrophobic doxorubicin

drug allows for pH sensitivity and therefore higher drug release in tumor cells where the pH is

lower. Other anionic pullulan derivatives prepared using γ-ray-irradiation have also been

reported70.

Figure 2.3: Chemical structure of carboxymethyl pullulan substituted at various positions

14

The use of CMP pullulan cross-linked hydrogels as antibacterial release wound dressings

was reported by Li56. Major characteristics needed for these applications include biocompatibility

and biodegradability because the polymer is in constant interaction with the wound bed. The

unique advantage of hydrogels is their ability to retain large quantities of aqueous medium

without dissolution of the polymer, which keeps the wound moist and prevents bacterial

infection. Creation of chemical crosslinks was explored initially using different linkages such as

ethylenediamine and dihydrazide, but ultimately cystamine-CMP hydrogels exhibited mechanical

properties superior to both. Cross-link density varied from 30 – 60%. Tensile strength of 1 mm

thick films ranged from 0.663 – 1.097 MPa with a swelling ratio of up to 4000%. Biocompatibility

tests showed no cytotoxicity and quick hemostatic ability prevented the accumulation of

exudates on the wound bed. Loading of the cystamine-CMP hydrogel with gentamycin sulfate,

an antibacterial agent, afforded the ability to suppress bacterial proliferation and protect against

bacteria. Drug load stayed constant at 45,000 U and in vitro studies showed fast release during

the initial 2 h followed by gradual release up to 40 h.

Sulfation has also been used to provide charge to the pullulan backbone58. Pullulan-based

nanoparticles were prepared for applications in transmucosal protein delivery57. Protein activity

is very susceptible to conformational changes in structure. Since they are very specific in their

actions, any minor change in structure can lead to decrease or elimination of function. Therefore,

new ways to deliver protein/protein drugs effectively and efficiently with better patient

compliance need to be addressed. In this study pullulan sulfate/chitosan (SP/CS) and pullulan

amine/κ-Carrageenan (AP/CRG) nanoparticles were made through polyelectrolyte complexation

with diameters of ≈ 250 nm and loading capacities of around 30%. Both nanoparticles were able

to associate with bovine serum albumin (BSA) protein and exhibited an absence of overt toxicity

towards a respiratory cell line (Calu-3). The investigators conducted in vitro studies which showed

that burst release is observed during the initial 2 h, then a steady state is achieved and no further

release is seen. Up to 30% of BSA was released, though it was not reported whether or not the

nanoparticles had any effect on the biofunctionality of the protein molecule itself.

Hydrophobically modified pullulan has also received a lot of attention recently43-44. To

reduce water solubility and increase organic solubility, esterified and etherified variations of

15

pullulan have been explored (Table 2.4). Their properties and solubility however greatly depend

on degree of substitution (DS) and molecular weight. Esterification of pullulan is usually carried

out in a base catalyzed reaction, typically with pyridine catalyst, and acid anhydride reagents.

However, Teramoto reported that esterification carried out under these conditions causes a

reduction in molecular weight, and opted for the use of acid chlorides instead, which gave

products that were reportedly higher in molecular weight35. The pullulan acetates they made

exhibited high glass transition temperatures (Tg) and decomposition temperatures (Td) and also

had tensile moduli on par with that of cellulose acetate with similar DS. Similarly, etherification

of pullulan can be carried out using a base (usually sodium hydroxide) catalyzed reaction, using

a haloalkane electrophile71. Ether bonds are more hydrolytically stable under practical usage

conditions, and therefore these derivatives can be used in a wider variety of applications.

Solvent Sample

Esters Ethers

PullAc

DS: 1.0

PullAc

DS: 2.4

PropylPull

DS: 1.02

PropylPull

DS: 2.45

ButylPull

DS: 1.28

ButylPull

DS: 2.61

Water Δ X X X X X

Ethyl Alcohol NR NR O O O O

Ethyl Acetate X Δ O O Δ Δ

Acetone X Δ X X X X

Chloroform X Δ X O X Δ

THF X Δ Δ O Δ O

DMSO O O O O O O

Toluene X X X Δ X Δ

Pyridine O O NR NR NR NR

O Soluble; Δ Partially Soluble; X Insoluble; NR Not Reported

Table 2.4: Table comparing the solubility of esterified and etherified pullulan with similar DS35,

71

16

Nishikawa reported that cholesterol-bearing pullulan, with its unique balance of

hydrophobicity and hydrophilicity, self-aggregates and forms stable nanoparticles with

hydrophobic cores72 (Figure 2.4). These nanoparticles were able to form complexes with α-

chymotrypsin (Chy) dimer with a radius of gyration of 12 nm at pH 4.2. Though it was found that

the helix content of Chy did change, when BSA was used to sustain release, the enzymatic activity

of the protein was not affected greatly. Cholesterol-grafted pullulan using vinyl ether grafts has

also been shown to produce nanogels, which can be complexed with small proteins such as BSA.

The gels showed unique pH sensitivity and an increase in the hydrodynamic radius of up to 135%

was observed when the pH shifted from 7.0 to 4.0. Labile (acL-CHP) and pH stable (acS-CHP)

analogs of the derivatives were prepared, with the intention of controlling release based on

degradation rate. The acid labile derivatives contain a cholesterol vinyl ether bond, which can

undergo rapid cleavage under acidic conditions whereas stable derivatives contained an ester

bonded cholesterol unit. As expected, the labile analog did show up to 80% degradation within

24 h at pH 4.0, which showed that release rate can be controlled.

17

Figure 2.4: Depiction of cholesterol modified pullulan (CH) and the structural differences of

labile (acL-CHP) and stable (acS-CHP) derivatives. Differences in structure are shown in red.

Adapted from Morimoto et. al.72

Long chain esters of pullulan were synthesized by reacting with carboxylic acids in the

presence of trifluoroacetic acid catalyst, to explore the use of pullulan as a plastic material73. The

products were reported as fully substituted by NMR methods and ranged from pullulan acetate

(2 carbon side chain) to pullulan myristate (14 carbons). As chain length increased, there was a

noticeable decline in glass transition temperature as well as tensile strength, but thermal stability

was increased. Solvent casting in chloroform and melt-pressing of alkyl pullulan esters readily

formed films, but only the acetate, propionate and butyrate derivatives were able to form

nanofibers due to the insolubility of the longer alkanoate polymers in hexafluoroisopropanol,

HFIP,the spinning solvent. Increasing chain length did cause an increase in elongation at break;

elongation values were higher than values reported for other polysaccharide esters.

Urethane pullulan derivatives have also been synthesized via reactions with isocyanate

compounds with DS ranging from 0.4 to 2.974. The derivatives showed good solubility in various

organic solvents such as DMSO, pyridine and chloroform. Solubility was influenced by DS and

higher substituted derivatives exhibited greater ranges of solubility. Isocyanates were also used

as crosslinking agents for pullulan/polyelectrolyte membranes for ion exchange75. However, the

cast membranes were impossible to peel off of the casting support.

Carbamate containing pullulan has been successfully prepared by reaction of 4-

nitrophenyl chloroformate to yield a carbonate derivative intermediate followed by slow addition

of a multifunctional amine (Figure 2.5)50. Amine functionality is of great interest for biomedical

applications given their ability to interact with bioactive systems via hydrogen bonding as well as

their cationic nature at low pH. Polyetheramine and poly(ethyl oxide)-poly(propyl oxide) amine

were conjugated with pullulan to make thermo and pH-sensitive block copolymers. Reductive

amination using end to end coupling of the aldehyde group on pullulan and the amino groups of

the amines76. Synthesis conditions did however affect the block length of pullulan chain.

18

Figure 2.5: Schematic of chloroformate activation to yield carbamate-containing derivatives.

Adapted from Bruneel50

Succinylated pullulan has also been synthesized via reaction with succinic anhydride in

the presence of the catalyst N,N’-dimethylaminopyridine77. Upon further conjugation with other

polymers or bioactive moieties, new drug delivery systems as well as site-specific targeting

vehicles can be achieved. Wang explored the possibilities of pullulan monosuccinate in a prodrug

formulation with cisplatin (CP-SUPA), via a coordination bond, for targeted therapy for

hepatocellular carcinoma (HCC) (Figure 2.6)78. Cisplatin is an inorganic complex depicted as

central platinum atom attached to chloride and ammonia atoms with each like pair in the cis

orientation to one another. It is used as a chemotherapeutic agent in a variety of clinical

treatments for various tumors. Due to a variety of possible delivery issues, inactive forms of a

drug, prodrugs, may be more suitable to be administered to patients, for example to minimize

discomfort or enhance bioavailability. CP-SUPA was shown to be very effective for inhibiting the

proliferation of HCC HepG2 cells. Additionally, due to pullulan’s tendency for high accumulation

in the liver and the enhanced permeability and retention effect (EPR), CP-SUPA was primarily

distributed in the liver and tumor after 24 h. The prodrug was shown to promote apoptosis and

arrest the cell cycle thereby inhibiting tumor growth. It also showed higher affinity for HepG2

cells rather than human lung epithelial A549 cells.

19

Figure 2.6: Synthesis of pullulan monosuccinate (SUPA) and the prodrug CP-SUPA via coordination, adapted from Wang et. al.78

The ability to control the positions of polysaccharide substituents (regioselectivity) has

been an important and challenging area of research79-80. Structure-property relationships of

polysaccharide derivatives are strongly impacted by regioselectivity, but polysaccharide

substituent regioselectivity is hard to control synthetically 81. Most regioselective syntheses of

polysaccharide derivatives involve a series of protecting and deprotecting steps with bulky

substituents in order produce the desired product. Some of these methods include tritylation,

silylation, halogenation and direct reaction with acyl moieties. The SN2 reaction of n-

bromosuccinimide (NBS) and triphenylphosphine (PPh3) with polysaccharides has been shown to

produce C-6 brominated derivatives with high regioselectivity82. Bromine can then undergo

nucleophilic substitution for further modification into other groups. Pereira reported the use of

this mechanism along followed by the use of Staudinger reduction chemistry to produce 6-amino

20

and 6-amido pullulan esters80 (Figure 2.7). These derivatives were shown contain amine or amido

substituents at only the C-6 position and exhibited a high DS of ester groups for each derivative

(5.9 – 7). The amido derivatives also were found to have high Tg values and a high tendency to

self-aggregate in solution.

Figure 2.7: Regioselective synthesis of 6-amino and 6-amido pullulan esters80

TEMPO mediated oxidation has also been found to be regioselective to the C-6 OH group,

allowing for the creation of a 6-carboxy polysaccharide83. The reaction is very specific for the C-6

21

position due to the accessibility of the primary hydroxyl group and because the cyclic

intermediate formed during this reaction would be too sterically hindered at other positions on

the backbone and cause a lot of ring strain. Initially, TEMPO is first converted to a nitrosonium

salt by sodium hypochlorite (NaClO) which then reacts with the primary hydroxyl group on the

pullulan forming the sterically hindered intermediate. The aldehyde is then formed which is then

converted to the carboxylic acid and converts sodium chlorite (NaClO2) to NaClO which can be

used to convert the reduced TEMPO to the nitrosonium salt to be reused in the reaction.

Figure 2.8: Oxidation of primary hydroxyl group using the 4-acetamido-TEMPO/NaClO/NaClO2 system adapted from Tamura et. al.83

Similar chemistry was performed on pullulan to yield 6-carboxypulluan ethers, which was

further modified to form amphiphilic pullulan ethers84 (Figure 2.9). The tetrabutylammonium 6-

carboxypullulan ethers that were synthesized by neutralization of 6-carboxypullulan to form the

organic-soluble tetrabutylammonium salt, followed by NaOH-catalyzed reaction with alkyl

halides, exhibited good solubility in organic solvents such as DMSO, DMF, MeOH AND EtOH.

22

Structure was confirmed by NMR and FTIR spectroscopy and some derivatives were found to

form micelles in aqueous media with very low critical micelle concentrations.

Figure 2.9: Synthesis of 6-carboxypullulan ethers via TEMPO oxidation

2.5 Drug Delivery

An important goal in polymeric research is the design of drug delivery vehicles that can

be used in formulations to be safely administered to patients85-86. Due to the increasing cost of

new drug development (many candidates fail due to delivery issues), as well as the difficulty in

finding vehicles to safely administer various therapies to patients, new cost effective and patient

complaint modes of drug delivery need to be explored. Most polymeric vehicles are designed to

enhance bioavailability, increase solubility, target specific sites, prolong release rate, increase

patient compliance, and/or greatly reduce side effects caused by these drugs. Bioavailability is

defined as the percentage of a drug dose that is able to enter circulation intact after it has been

administered. The four main modes of drug delivery are intravenous, inhalation, transdermal and

oral.

The first and most efficient of these methods of delivery is intravenous injection (IV).

Intravenously delivered drug provides the highest bioavailability and very precise dosages can be

delivered. The bioavailability of drug administered through IV is 100% because the entire dosage

can be directly put into circulation87. This provides immediate therapeutic affect to the by patient

http://www.sciencedirect.com/science/article/pii/S0144861712012520#gr1

23

and is the most efficient way to give medicine during surgery and to incapacitated patients during

hospital stays. One of the issues with this method is that crystalline hydrophobic drugs that have

poor solubility cannot be administered. This is due to the possibility of recrystallization after

injection, which can lead to sepsis and death. There is also the difficulty of getting them in

solution, as some drugs can have solubilities of a few μg per mL or less. One of the major

drawbacks is the low patient compliance, which can lead to irregular administration and cause

serious health problems. Other issues that arise with IV injection include formation of emboli,

infection, pain, infiltration (medication enters surrounding tissue instead of vein) and phlebitis

(inflammation of vein). Subcutaneous injection does offer the same benefits as IV, but safety

concerns and lack of patient compliance also cause irregularity in its use.

Inhalation is another method that does not evoke the safety concerns attributed to IV

injection. Drugs used in this form of administration are aerolized and inhaled by the patient,

entering the respiratory tract to eventually be absorbed into the body through the alveoli. Large

particles usually deposit in the upper respiratory tract by impaction which is a usually a problem

for patients not trained in using the devices. In order to be effective, the particles have to be

small enough (0.5 – 5 μm) to avoid getting stuck in the upper respiratory tract88. One advantage

is that uptake though the alveolus is faster than though the skin or GI tract and therefore

therapeutic effects can be felt much faster. In addition, since the target organ of most of these

formulations is the lungs, transport to target site is immediate. Drug therapies delivered via this

mode usually are for treatment of diseases such as asthma, cystic fibrosis and chronic obstructive

pulmonary disease (COPD). Some of the issues that plague this mode of delivery stem from the

inability of some drugs to be cleared from the lungs. Blockage of the alveoli due to residues or

impurities can lead to decreases in oxygen uptake as well as cause permanent damage to the

respiratory system. In addition, because only small molecules that can be aerolized can be used

in this application, the range of drugs that can be delivered by this method is limited.

Transdermal administration is a very patient compliant method for drug delivery. This

method utilizes passive permeation of the drug through the skin to allow for a slow absorption

over an extended period. Nicotine (smoking cessation) and scopolamine (seasickness) patches

are two of the applications in commercial use. However, very few drugs are able to permeate

24

passively though the skin due to their chemical nature as well as the skin’s defenses to keep

foreign molecules out. The skin is the body’s first line of defense and serves many key roles. It is

composed of the epidermis, dermis and hypodermis, all which work together to keep foreign

substances from getting into the body. Most drugs for this method are chosen based on their

solubility and diffusivity in the stratum corneum, the outer most layer of the epidermis, and that

is directly related to melting point (MP < 250 oC) and molecular weight (MW < 500 kDa)89.

Additionally, the drug also has to show moderate lipophilic characteristics (log P range from 1-5)

to be able to dissolve in the skin. Even drugs that do meet these criteria have to be able to achieve

and maintain therapeutic plasma levels, otherwise the effects of the drug will not be felt by the

patient or multiple patches would be required over a short period. There are technologies being

developed which uses micro needles as a way to bypass the epidermis and dissolve into the skin

to facilitate better absorption90. These do show promise to expand the amount of drugs that can

be delivered transdermally, but commercially only a few drugs can be administered via this mode

of delivery.

The most patient complaint mode of delivery is oral administration. Drugs that utilize this

mode of delivery can be taken in many forms such as pills, capsules and liquids. Oral

administration allows for precision in the amount of medication administered (although not

necessarily in the amount that reaches the bloodstream). Most drugs are primarily absorbed in

the small intestines of the GI tract, which means that they have to pass first through the harsh

environment of the stomach91. This is one of the major drawbacks of oral administration and can

cause degradation of the drug as well as conformational changes in protein drugs, for example,

which can render the drug partially or entirely inactive. In addition, drugs that make it through

the stomach still face the problem of being bioavailable to be absorbed through the villi. This is

due to the highly hydrophobic nature of the epithelial layer of the GI tract, which makes it

extremely difficult for drugs pass through and get into circulation. This leads to pharmaceutical

companies using large drug dosages only to have a small amount actually make it into the blood

stream, which causes high drug costs, imprecision, variability of delivered dosage, wasting of

drugs, and in some cases more severe or frequent side effects. Additionally, since most drugs are

crystalline and hydrophobic, when they enter the GI tract bioavailability can decrease further due

25

to drug recrystallization. Drugs that do eventually get through the epithelium face the danger of

first pass metabolism, in which they can be metabolized by the liver before they can get into

circulation.

A lot of research has been done on trying to overcome many of the pitfalls in each of

these areas of drug delivery. In particular, the use of amorphous solid dispersions (ASDs) for orally

delivered drugs has shown promise in addressing problems faced due to the harsh environment

in the GI tract as well as the crystalline and hydrophobic nature of most drugs9, 92-93. ASDs operate

by trapping the drug in a molecular dispersion, in an amorphous polymer matrix, to prevent drug

recrystallization. Frequently ASD polymers contain pH-sensitive groups, providing a mechanism

for release in the neutral media of the intestines. These formulations lead to supersaturation of

the drug in the intestines, and increase in apparent solubility, which increases bioavailability due

both to enhanced drug solubility and to enhanced drug absorption. To be effective as an ASD

matrix, there are a few requirements that polymers must meet. The polymer and the product

after decomposition must be non-toxic so as to not invoke immune responses or cause other

health problems. Miscibility with the hydrophobic drug must also be possible in order to break

up crystallinity and suspend the drug in the polymer matrix. A high Tg is also desirable to keep

the formulation Tg well above any practical ambient temperature, thereby stopping drug

migration and recrystallization, even under conditions of high ambient temperature and

humidity, and even when the drug turns out to be a plasticizer, as they sometimes do. In order

to facilitate release in the GI tract, a pH-sensitive group should be attached to allow for swelling

and releasing of the drug. During this time, it is very possible for recrystallization to occur, so the

polymer also has to have some slight water solubility so that it can associate with dissolved drug,

and thereby prevent crystallization.

In industry, the two main polymers used for ASD formulations are

poly(vinylpyrrolidinone)-vinyl acetate (PVP-VA) and hydroxypropyl methyl cellulose acetate

succinate (HPMCAS). These formulations show a lot of promise in raising apparent solubility,

enhancing oral bioavailability and increasing dissolution rate with respect to just the crystalline

drug. They also help in addressing the issues faced with enduring the harsh conditions of the GI

tract, which could ultimately lead to lower dosages and cheaper medication. However, PVP-VA

26

due to high water-solubility, affects the stability of the formulation by lowering Tg and increasing

molecular mobility, thereby causing premature release of drug and recrystallization in the

matrix94. HPMCAS does not have this issue, but due to a complex synthetic route, issues with

controlling four different substituents along the cellulose backbone and the slower release profile

of more hydrophobic polymers, there is a need for new systems tailored towards ASD standards.

Cellulose ω-carboxyalkanoates have been shown to be very promising as new ASD polymers

because they have been shown to retard recrystallization of the anti-HIV drug ritonavir and

antibacterial clarithromycin95-97. They also exhibit high glass transition temperatures and are non-

toxic. However, these derivatives do suffer from poor water solubility, which causes limitations.

Natural pullulan does exhibit water solubility, and after chemical modification to produce ω-

carboxyalkanoate derivatives, they will have more enhanced solubility than their cellulose

counterparts. Stronger polymer-drug interactions in solution would be possible and would give

rise to a more effective ASD matrix. These pullulan derivatives may also be able to utilize other

modes of drug delivery, such as intravenous administration, due to pullulan’s ability to be cleared

from circulation, which cellulosic polymers lack.

2.6 Conclusion

The use of chemically modified pullulan for biomedical applications has been a popular

topic of research. These derivatives have proven not only to be effective, but because of the non-

toxic, biodegradable and non-mutagenic properties of pullulan, they can be adapted for a variety

of other applications as well. Particularly in drug delivery, research has been geared towards

utilizing the liver accumulating tendency of pullulan through intravenous injections as a drug

targeting system. These systems show high accumulation in the liver and are promising as new

ways to combat a variety of liver carcinomas, blastomas and sarcomas while reducing the side

effects of these potent drugs on healthy cells.

Despite the significant body of literature on pullulan and its use in intravenous drug

delivery systems, there is very little published on its capability for oral drug delivery, in particular

27

for amorphous solid dispersions. Previous literature on cellulose ω-carboxyalkanoates has shown

that polysaccharides do possess the ability to make successful ASD polymers. Additionally, the

substituents used were shown to have a tremendous effect on increasing miscibility with

hydrophobic drugs and providing polymer-drug interactions to prevent recrystallization and

provide site-specific release. The potential of pullulan derivatives with similar modification would

produce a new set of polymers, advantaged by superior water solubility vs. their cellulose

counterparts, geared towards oral drug delivery, but with applications for intravenous or other

modes of administration.

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38

CHAPTER 3: Synthesis of Pullulan ω-carboxyalkanoates for Drug

Nanodispersions

3.1 Abstract

Polysaccharide modification to increase application and functionality has shown

particular promise for drug delivery systems. The use of amorphous solid dispersions (ASDs) has

proven to be an effective way to increase apparent solubility and bioavailability of crystalline,

hydrophobic drugs in the gastrointestinal (GI) tract. Synthesis of cellulose ω-carboxyalkanoates

has already been reported and these polymers show extremely promising results as ASD

matrices; they are however limited in some cases due to their modest water solubility. Natural

pullulan however is water-soluble and modification with ω-carboxyalkanoates is expected to give

rise to derivatives with improved solubility vs. cellulosic counterparts, also possessing the ability

for strong drug-polymer interactions in solution. We herein report the successful synthesis of

pullulan ω-carboxyalkanoates for applications in drug delivery. These derivatives exhibited good

solubility in organic solvents such as DMSO, acetone (DMK) and THF. Glass transition

temperatures (Tg) of these derivatives peaked to 93 oC at pullulan suberate acetate (DSω-carboxy =

0.51) and all exhibited high decomposition temperatures (Td) above 300 oC. Calculated solubility

parameters (SP) ranged from 21.48 to 23.32 MPa1/2. The pullulan suberate ω-carboxyalkanoate

derivatives due to their good thermal properties and ideal solubility parameters have strong

potential as candidates for ASD polymer formulations.

3.2 Introduction

Pullulan is a bacterial polysaccharide secreted from the black yeast-like fungus

Aureobasidium pullulans 1-2. It is a linear exopolysaccharide comprised entirely of the

monosaccharide glucose 3. Structurally, pullulan is depicted as having the trisaccharide repeat

unit maltotriose, connected by -16 linkages. The result is a polymer containing 2 α-(14)

39

linkages for each 1 α-(16) linkage; the repeat unit can be represented as [4)-α-D-Glcp-(16)-

α-D-Glcp-(14)-α-D-Glcp-(1]. Unlike the more industrially utilized glucan cellulose, pullulan is

water soluble, biodegradable, and thus may be cleared from circulation 4. In addition, its low

toxicity and lack of immunogenicity has made it very useful in the food industry in the capacity

as an adhesive, binder and food thickener 5-6. Due to its excellent oxygen barrier properties and

film forming capability, pullulan has also found use in packaging applications worldwide 7-8.

Chemical modification of pullulan may permit property adjustment to enhance processability

and/or match performance criteria, in order to capitalize on the innate positive pullulan

properties enumerated above. This concept is illustrated by published studies using modified

pullulans in drug delivery 9, as anticoagulants 10, for wound healing 11, and in gene delivery 12.

Though water solubility is a property of pullulan that is frequently an asset to a particular

application, in other cases water solubility may be a drawback, so chemical modification has

frequently targeted reduction in water solubility, while imparting structural charges or adding

functional groups that adjust properties to those needed for specific end uses 13-15.

Addition of carboxyl groups to polymers has often been found to be useful for drug

delivery applications. The pH-responsive carboxyl group may enable controlled release of a drug

load through polymer swelling at neutral pH, as well as providing site specific targeting 16-17. For

example, in order for protein-digesting enzymes to function efficiently in the stomach, a low pH

is required. This is primarily achieved by the secretion of gastric acid from parietal cells, which

gives the stomach an overall pH of ≈ 1.5-3.5. In these conditions, carboxyl groups are protonated

and the drug load stays restricted and protected in the polymer matrix 18. However when the

contents on the stomach are released into the small intestine, the pH increases to near neutral,

which in turn causes the formation of carboxylate anions. Due to electrostatic repulsions of the

now negatively charged groups, the polymer matrix expands and facilitates the release of the

drug load into the small intestine, from which the drug can be absorbed into the circulation.

Synthesis of amphiphilic polymers containing both hydrophobic and hydrophilic groups

can be beneficial in the preparation of amorphous solid dispersions (ASD) of hydrophobic,

crystalline drugs. These ASDs work by dispersing the drug molecularly in a polymer matrix,

creating a metastable amorphous form of the drug that is kept in this high energy form by a

40

combination of high polymer Tg (preventing drug mobility even at high ambient levels of

temperature and humidity) and by promoting drug/polymer miscibility through intermolecular

forces 19. By eliminating drug crystallinity through molecular dispersion in the polymer matrix,

the apparent solubility of the drug is increased, and thereby the enterocytes of the

gastrointestinal (GI) tract are exposed to higher drug concentrations, resulting in higher chemical

potentials across the epithelium, in turn resulting in higher levels of permeation and more

complete drug bioavailability. Carboxy-containing polymers are very promising due to their

ability to promote pH controlled release in the GI tract as well as their ability to increase

miscibility with the drug in the polymer matrix through interaction with the amine and hydroxyl

groups that most drugs contain.

Syntheses of ω-carboxyalkanoate cellulose esters containing adipate, suberate and

sebacate groups have recently been reported 20-21. These derivatives have shown great promise

as ASD polymers, as their combinations of high Tg, carboxyl content, amphiphilic nature, and

solubility parameters promote increased miscibility and dispersion stability with hydrophobic

drugs. The carboxyl group ionizes in the stomach and swells in the neutral environment of the

intestines to provide a mechanism for drug release 22. Furthermore, studies have shown that

cellulose acetate adipate propionate (CAAdP) strongly inhibits crystal growth of ritonavir, a drug

used in multiple marketed formulations that combat the human immunodeficiency virus (HIV) 23.

Retardation of drug crystallization after release and prior to absorption is an important

performance criterion for ASD polymers, to facilitate drug absorption into the blood stream

through the enterocytes that line the small intestines 9.

Esterification of pullulan has been previously reported 24-25. Short chain esters such as

acetate, propionate and butyrate are usually prepared by reaction with carboxylic acid

anhydrides in the presence of a base such as pyridine or triethylamine (TEA). Esterification with

-carboxylalkanoyl moieties is more complicated, since esterification with a diacid or derivative

activated at both carbonyls would be certain to afford cross-linked, insoluble products. In related

work investigating synthesis of cellulose -carboxyalkanoates, reaction of cellulose with the

cyclic adipic anhydride was investigated 26. Reaction with the anhydride did indeed occur

instantly, but cross-linking and gelation were also observed. Mechanistic investigations showed

41

that poly(adipic anhydride) impurities in the adipic anhydride reacted in more than one location

on the same poly(anhydride) chain with cellulosic hydroxyls, causing crosslinking, gelation and

the observed insoluble product. In order to avoid this undesired crosslinking, Kar, et al. 21 took a

different approach, involving a mono-activated, mono-protected synthon of the diacid. They

prepared the monobenzyl ester of adipic acid, and then converted it into the monofunctional

monobenzyl adipoyl chloride. Reactions of this monofunctional reagent with cellulose and its

ester derivatives afforded soluble benzyl cellulose -carboxyalkanoate esters, containing no

cross-linked product as evidenced by spectroscopy and product solubility. The benzyl groups

were readily removed by hydrogenolysis, affording a variety of cellulose -carboxyalkanoates

containing other ester groups, which had good organic solubility, high Tg values, and a small

amount of solubility in neutral water. Several of these cellulose -carboxyalkanoates provided

excellent performance in ASDs 27.

Polymers currently in use for ASD formulations are restricted to a small set due to the

strict performance requirements for ASD polymers22-23. Due to the high cost and long duration of

new polymer approvals, many current ASDs contain polymers that have been chosen from

already approved pharmaceutical formulations and are not designed specifically for use in ASDs.

As a result, there is an increasing need for new polymers designed for use in ASD formulations.

Two polymers commonly used in ASDs are poly(vinylpyrrolidinone)/ vinyl acetate (PVP-VA) and

hydroxypropyl methyl cellulose acetate succinate (HMPCAS), but they are not without their

problems. PVP-VA has quite high water-solubility, especially in the low pH environment of the

stomach, which can cause premature release of drug.28. HPMCAS does not have this issue, but

due to the complex synthetic route and issues with analyzing and controlling four different

substituents, other candidates have to be explored. Pullulan ω-carboxyalkanoates are interesting

candidates because they specifically address all of the parameters (high Tg, miscibility, slight

water solubility, pH-sensitivity) for making a good ASD polymer. Beyond oral use, these pullulan

derivatives may be adaptable for other modes of delivery. Pullulan has been shown to be

completely biodegradable in the human body and its byproducts can be cleared without invoking

an immune response however, its derivatives have not yet been studied. In addition, pullulan has

been shown to orient in a random coil conformation when in solution primarily due to the α-

42

(16) linkage, which imparts flexibility to the backbone 29. Flexibility promotes better dispersion

of drug molecules in the polymer matrix, which can increase drug loading and better prevent

recrystallization. These useful properties open the possibilities of other modes of drug delivery

such as intravenous and inhalation administration.

Herein we explore methodologies for synthesis of pullulan ω-carboxyalkanoates, and

evaluation of these polymers with regard to fit of their properties with requirements for

amorphous solid dispersion and other biomedical applications. We hypothesize that synthesis of

these derivatives will permit creation of nanoscale homogeneous dispersions of drugs in pullulan

-carboxyalkanoate matrices. We further hypothesize that these nanodispersions will generate

supersaturated drug concentrations, and that nanoparticulate versions of these nanodispersions

may be highly valuable not only for oral drug administration, but for intravenous, inhalation, and

other modes of administration where there may be concerns about the ability to clear cellulosic

polymers from the body.

Figure 3.1: Synthetic scheme for pullulan ω-carboxyalkanoates.

3.3 Experimental

43

3.3.1 Materials

Pullulan JP, USP-NF (Mw = 450 kDa, Mn = 200 kDa) was obtained from the Hayashibara

Company and was dried overnight under vacuum at 115oC prior to use. N,N-Dimethylacetamide

(DMAc, Fisher) was dried over 4Å molecular sieves before use. Triethylamine (TEA, 99% Acros

Organics), acetic anhydride (99+%, Acros Organics), propionic anhydride (97%, Aldrich) and n-

butyric anhydride (98%, Acros Organics) were used as provided. Adipic acid (99%, Acros

Organics), suberic acid (99%, Acros Organics), sebacic acid (98%, Acros Organics), oxalyl chloride

(98%, Acros Organics) and p-toluenesulfonic acid monohydrate (PTSA, 99% extra pure, Acros

Organics) were used as provided. Toluene (HPLC Grade, Fisher), benzyl alcohol (Aldrich),

methylene chloride (DCM, HPLC Grade, Fisher), N,N-dimethylformamide (DMF, Fisher) and

tetrahydrofuran (THF, stabilized with 0.025% BHT, Spectrum) were used as provided.

Hydrochloric acid (Fisher), sodium hydroxide (Fisher), hexanes (Fisher), sodium bicarbonate

(NaHCO3, Reagent Grade, Fisher) and chloroform (Fisher) were used as provided. Chloroform-d

(99.8 atom% D) and dimethyl sulfoxide-d6 (99.9 atom% D) for NMR were acquired from

Cambridge Isotope Laboratories, Inc. and Pd(OH)2/C for hydrogenolysis was obtained from

Sigma-Aldrich.

3.3.2 Measurements

NMR samples were prepared by dissolving 20-25 mg (for 1H) or 50 mg (for 13C) of polymer

in 0.7 mL of DMSO-d6 or CDCl3 and pipetted into standard 5 mm NMR tubes. Spectra were

obtained on Bruker Avance 500 spectrometer using 16-32 scans for 1H samples and a minimum

of 7500 scans for 13C samples. Chemical shifts are reported relative to the solvent.

Differential scanning calorimetry (DSC) analysis of polymers was performed on a TA

Discovery DSC. The polymer (5 mg) was loaded in Tzero aluminum pans and equilibrated at 25oC.

Samples were heated to 80oC at 20oC/min, cooled to -20oC at 100oC/min and heated again to

220oC at 15oC/min. The glass transition temperature (Tg) was recorded as the inflection point and

was determined from the second heat scans.

Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy was

conducted using a Thermo Electron Nicolet 8700 instrument in transmission mode. The samples

44

were placed on the crystal and a screw was used to press the sample into a flat disk. One hundred

and twenty eight (128) scans were used to obtain spectra.

Solubility was determined by placing 5 mg of polymer into 1 mL of solvent under vortex

mixing for 5-10 min at room temperature. A visual exam was used to determine solubility.

Thermo Gravimetric Analysis (TGA) was obtained on a TGA Q500. The polymer (10 mg)

was loaded onto the sample pan and heated to 600oC at 10oC/min. The decomposition

temperature (Td) was reported as the temperature at 50% weight loss.

Solubility parameters (SP) were calculated using a method based only on the knowledge

of the polymer structure. The method was proposed by Fedors30 and used as a comparison of the

relative hydrophobicities of the polymers. It is based on group additive constants and the

contribution of a large number of functional groups. The formula used for the evaluation is as

follows:

𝛿 = √Σ𝑖Δ𝑒𝑖

Σ𝑖Δ𝑣𝑖= √

ΔΕ𝑣

𝑉

where Δ𝑒𝑖 and Δ𝑣𝑖 are the additive atomic and group contribution for the energy of vaporization

and molar volume, respectively. For polymers with higher molecular weight that have a Tg higher

than 25 oC, there is a deviation between the experimentally measured ΔΕ𝑣 and 𝑉 and the

estimated values. To take into account the divergence in the 𝑉 values, a small correlation factor

was introduced and taken into account :

Δ𝑣𝑖 = 4𝑛, 𝑛 < 3

Δ𝑣𝑖 = 2𝑛, 𝑛 ≥ 3

where 𝑛 is the number of the main chain skeletal atoms in the smallest repeating unit of the

polymer.

45

The degree of substitution (DS) values of pullulan esters are described per

anhydroglucose unit (AGU), with a maximum overall DS of approximately 3. These values were

obtained through 1H NMR spectroscopy. The DS of the ω–carboxyester group (DScarboxyester) was

calculated using the following formula:

𝐷𝑆carboxyester = 7𝐼𝑝ℎ𝑒𝑛𝑦𝑙

5𝐼𝑏𝑎𝑐𝑘𝑏𝑜𝑛𝑒+𝑏𝑒𝑛𝑧𝑦𝑙𝑖𝑐 ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛 − 2𝐼𝑝ℎ𝑒𝑛𝑦𝑙

The phenyl peak was observed at 7.26 ppm and the pullulan backbone was seen in the

region of 3.4 – 5.5 ppm. Calculation of the DS of the ester groups (DSester) was performed using

the formula:

𝐷𝑆𝑒𝑠𝑡𝑒𝑟 =10𝐴

(3𝐶 + 𝐴)

DS values for the esters were obtained after hydrogenolysis and incorporated the

integration of the methyl protons (A) at 1.8 – 2.2 ppm for acetyl, 0.8 – 1.1 ppm for propyl and 0.7

– 0.9 ppm for butyl with respect to the pullulan backbone (C) which encompassed the 3.4 – 5.5

ppm region.

3.3.3 Synthesis

3.3.3.1 Synthesis of Monobenzyl Suberate

The method for this synthesis was adapted from Liu, et al. 22. Suberic acid (0.5 mol, 87.10

g), PTSA (5 mmol, 0.97 g), toluene (400 mL) and benzyl alcohol (0.6 mol, 62.2 mL) were added to

a round bottom flask and a Dean-Stark trap apparatus was attached. The mixture was allowed to

reflux until 10.8 mL of water (0.6 mol) was collected which indicated reaction completion. The

mixture was allowed to cool to room temperature before adding 300 mL of DI water under

magnetic stirring. The pH of the mixture was adjusted to 9.0 using 6 M NaOH. Using a separatory

funnel, the aqueous layer was collected and washed with 100 mL of diethyl ether and

subsequently recollected. Ethyl ether (400 mL) was added to the aqueous layer under magnetic

stirring and the pH was adjusted to 2.0. After separation and collection of the organic layer, it

46

was washed with 200 mL of 1 M NaHCO3 and concentrated under reduced pressure. The colorless

oil was vacuum-dried for 1 h and the final product, a white powder, was collected. Yield 19.0%:

1H NMR (CDCl3): 1.34 (m, 4H), 1.63 (m, 4H), 2.34 (m, 4H), 5.11 (s, 2H), 7.35 (m, 5H).

A similar procedure was used for the synthesis of monobenzyl adipate (colorless oil). Yield

15.0%: 1H NMR (CDCl3): 1.72 (m, 4H), 2.39 (m, 4H), 5.14 (s, 2H), 7.37 (m, 5H).

A similar procedure was used for the synthesis of monobenzyl sebacate (white powder).

Yield 24.0%: 1H NMR (CDCl3): 1.29 (s, 8H), 1.62 (m, 4H), 2.33 (m, 4H), 5.11 (s, 2H), 7.35 (m, 5H).

3.3.3.2 Synthesis of Monobenzyl Suberoyl Chloride

The method for this synthesis was adapted from Liu, et al. 22. Monobenzyl suberate (0.051

mol, 13.53 g), 300 mL of DCM, and 5 drops of DMF were placed in a round-bottom flask. The

solution was stirred magnetically in an ice bath at 0oC until the ester completely dissolved. The

ice bath was removed after dropwise addition of the oxalyl chloride (24.42 mL, 5.6 eq). Stirring

was continued until gas evolution stopped indicating the end of the reaction. After the solution

was concentrated under reduced pressure, 30 mL of toluene was added and the product was

concentrated again. The yellow oil product was filtered and used as is. Yield 93.0%: 1H NMR

(CDCl3): 1.34 (m, 4H), 1.69 (m, 4H), 2.34 (m, 2H), 2.85 (m, 2H), 5.11 (s, 2H), 7.1-7.4 (m, 5H).

A similar procedure was used for the synthesis of monobenzyl adipoyl chloride (yellow

oil). Yield 95.0%: 1H NMR (CDCl3): 1.71 (m, 4H), 2.35 (m, 2H), 2.89 (m, 2H) 5.14 (s, 2H), 7.1-7.4

(m, 5H).

A similar procedure was used for the synthesis of monobenzyl sebacoyl chloride (yellow

oil). Yield 95.0%: 1H NMR (CDCl3): 1.30 (s, 8H), 1.69 (m, 4H), 2.35 (m, 2H), 2.87 (m, 2H), 5.11 (s,

2H), 7.35 (m, 5H).

3.3.3.3 Synthesis of Monobenzyl Pullulan Adipate Acetate/Propionate/Butyrate

47

Pullulan (5.5 mmol AGU, 1 g) was dissolved in 50 mL of DMAc under magnetic stirring at

80oC in a round-bottom flask. Triethylamine (18.4 mmol, 3.3 eq AGU, 2.5 mL) followed by

monobenzyl adipoyl chloride (16.4 mmol, 3 eq AGU, 4.2 g) were added via syringe. After stirring

for 23 h at 80oC, triethylamine (33.0 mmol, 6 eq AGU, 4.1 mL) followed by either acetic anhydride

(22.0 mmol, 4 eq/AGU, 2.07 mL), propionic anhydride (22.0 mmol, 4 eq/AGU, 2.8 mL) or n-butryic

anhydride (22.0 mmol, 4 eq/AGU, 3.55 mL) were added and allowed to react for an additional 10

h at 80oC. After the reaction was completed, the mixture was filtered to remove the triethylamine

hydrochloride precipitate and the solution was dialyzed against water for 3 days, changing the

water daily. The precipitate was dried in a vacuum oven overnight at 40oC after which it was

redissolved in chloroform (~ 80 mL) and re-precipitated in hexanes (~ 400 mL). The final product

was then collected by filtration and dried in a vacuum oven at 45oC.

For the preparation of the lower DS pullulan adipate esters, monobenzyl adipoyl chloride

(10.9 mmol, 2 eq/AGU, 2.8 g) and triethylamine (12.27 mmol, 2.2 eq/AGU, 1.67 mL) were reacted

with (5.5 mmol AGU, 1 g) pullulan. The rest of the procedure was carried out as described above.

Monobenzyl Pullulan Adipate Acetate Yield: 84.3% (1.56 g, 4.6 mmol). 1H NMR (CDCl3):

1.8-2.2 (COCH3 of acetate), 1.4-1.6 (COCH2CH2CH2CH2CO of adipate), 2.32 (COCH2CH2CH2CH2CO

of adipate), 3.4-5.5 (pullulan backbone), 5.03 (CH2C6H5), 7.27 (CH2C6H5).

Monobenzyl Pullulan Adipate Propionate Yield: 90.2% (1.76 g, 5.0 mmol). 1H NMR (CDCl3):

1.05 (COCH2CH3 of propionate), 1.6 (COCH2CH2CH2CH2CO of adipate), 2.2-2.5

(COCH2CH2CH2CH2CO of adipate, COCH2CH3 of propionate), 3.4-5.5 (pullulan backbone), 5.03

(CH2C6H5), 7.27 (CH2C6H5).

Monobenzyl Pullulan Adipate Butyrate Yield: 88.3% (1.73 g, 4.9 mmol). 1H NMR (CDCl3):

0.8-0.9 (COCH2CH2CH3 of butyrate), 1.59 (COCH2CH2CH2CH2CO of adipate, COCH2CH2CH3 of

butyrate), 2.30 (COCH2CH2CH2CH2CO of adipate, COCH2CH2CH3 of butyrate), 3.4-5.5 (pullulan

backbone), 5.03 (CH2C6H5), 7.27 (CH2C6H5).

3.3.3.4 Synthesis of Monobenzyl Pullulan Suberate Acetate/Propionate/Butyrate

48

A procedure similar to that used for the synthesis of the monobenzyl pullulan adipate

esters was employed. Pullulan (5.5 mmol AGU, 1 g) was reacted with triethylamine (18.4 mmol,

3.3 eq/AGU, 2.5 mL) and monobenzyl suberoyl chloride (16.4 mmol, 3 eq/AGU, 4.66 g). The rest

of the procedure was carried out as described earlier.

For the preparation of the lower DS pullulan suberate esters, monobenzyl suberoyl

chloride (10.9 mmol, 2 eq/AGU, 3.11 g) and triethylamine (12.27 mmol, 2.2 eq/AGU, 1.67 mL)

were reacted with (5.5 mmol AGU, 1 g) pullulan. The rest of the procedure was carried out as

described.

Monobenzyl Pullulan Suberate Acetate Yield: 93.3% (2.24 g, 5.1 mmol). 1H NMR (CDCl3):

1.8-2.2 (COCH3 of acetate), 1.25 (COCH2CH2CH2CH2CH2CH2CO of suberate), 1.55

(COCH2CH2CH2CH2CH2CH2CO of suberate), 2.25 (COCH2 CH2CH2CH2 CH2CH2CO of suberate), 3.4-

5.5 (pullulan backbone), 5.03 (CH2C6H5), 7.27 (CH2C6H5).

Monobenzyl Pullulan Suberate Acetate Yield: 74.1% (1.86 g, 4.1 mmol). 1H NMR (CDCl3):

1.03 (COCH2CH3 of propionate), 1.25 (COCH2CH2CH2CH2CH2CH2CO of suberate), 1.55

(COCH2CH2CH2CH2CH2CH2CO of suberate), 2.25 (COCH2CH2CH2CH2CH2CH2CO of suberate,

COCH2CH3 of propionate), 3.4-5.5 (pullulan backbone), 5.03 (CH2C6H5), 7.27 (CH2C6H5).

Monobenzyl Pullulan Suberate Butyrate Yield: 78.5% (1.97 g, 4.3 mmol). 1H NMR (CDCl3):

0.7-0.9 (COCH2CH2CH3 of butyrate), 1.24 (COCH2CH2CH2CH2CH2CH2CO of suberate), 1.59

(COCH2CH2CH2CH2CH2CH2CO of suberate, COCH2CH2CH3 of butyrate), 2.30

(COCH2CH2CH2CH2CH2CH2CO of suberate, COCH2CH2CH3 of butyrate), 3.4-5.5 (pullulan


3.3.3.5 Synthesis of Monobenzyl Pullulan Sebacate Acetate/Propionate/Butyrate

A procedure similar to that used for the synthesis of the monobenzyl pullulan adipate

esters was employed. Pullulan (5.5 mmol AGU, 1 g) was reacted with triethylamine (18.4 mmol,

3.3 eq/AGU, 2.5 mL) and monobenzyl sebacoyl chloride (16.4 mmol, 3 eq/AGU, 5.13 g). The rest

of the procedure was carried out as previously described.

49

For the preparation of the lower DS pullulan sebacate esters, monobenzyl sebacoyl

chloride (10.9 mmol, 2 eq/AGU, 3.42 g) and triethylamine (12.27 mmol, 2.2 eq/AGU, 1.67 mL)

were reacted with (5.5 mmol AGU, 1 g) pullulan. The rest of the procedure was carried out as

previously described for the adipates.

Monobenzyl Pullulan Sebacate Acetate Yield: 97.3% (2.17 g, 5.4 mmol). 1H NMR (CDCl3):

1.8-2.2 (COCH3 of acetate), 1.21 (COCH2CH2CH2CH2CH2CH2CH2CH2CO of sebacate), 1.54

(COCH2CH2CH2CH2CH2CH2CH2CH2CO of sebacate), 2.27 (COCH2CH2CH2CH2CH2CH2CH2CH2CO of

suberate), 3.4-5.5 (pullulan backbone), 5.03 (CH2C6H5), 7.27 (CH2C6H5).

Monobenzyl Pullulan Sebacate Propionate Yield: 74.5% (1.75 g, 4.1 mmol). 1H NMR

(CDCl3): 1.06 (COCH2CH3 of propionate), 1.21 (COCH2CH2CH2CH2CH2CH2CH2CH2CO of sebacate),

1.54 (COCH2CH2CH2CH2CH2CH2CH2CH2CO of sebacate), 2.25 (COCH2CH2CH2CH2CH2CH2CH2CH2CO

of suberate, COCH2CH3 of propionate), 3.4-5.5 (pullulan backbone), 5.03 (CH2C6H5), 7.27

(CH2C6H5).

Monobenzyl Pullulan Sebacate Butyrate Yield: 94% (2.2 g, 5.2 mmol). 1H NMR (CDCl3): 0.7-

0.9 (COCH2CH2CH3 of butyrate), 1.20 (COCH2CH2CH2CH2CH2CH2CH2CH2CO of sebacate), 1.54

(COCH2CH2CH2CH2CH2CH2CH2CH2CO of sebacate, COCH2CH2CH3 of butyrate), 2.26

(COCH2CH2CH2CH2CH2CH2CH2CH2CO of suberate, COCH2CH2CH3 of butyrate), 3.4-5.5 (pullulan


3.3.3.6 Hydrogenolysis of Monobenzyl Pullulan Suberate Acetate

In a round bottom flask, monobenzyl pullulan suberate acetate (0.5 g) was dissolved in

150 mL of THF and 500 mg of palladium hydroxide on carbon, 20 wt. % loading (dry basis), was

added to the solution followed by triethylsilane (18.6 mmol, 2.98 mL, 10 eq/benzyl group). A

balloon connected to a 3-way valve was attached to the flask and the mixture was placed under

vacuum under stirring. After there were no more bubbles left into solution, the valve was closed

and nitrogen was attached to the valve. The balloon and flask were then simultaneously filled

with nitrogen. The vacuum was then reattached, only opened to the flask. Evacuation followed

50

by a purge with nitrogen in the flask was done three times followed by a refill of the balloon with

hydrogen. The valve was left open only to the flask and balloon and hydrogenolysis was carried

out for 24 h at room temperature. The mixture was then filtered through Celite™ and the solution

once again went through the previous procedure and left for 24 h. After that, the mixture was

filtered through Celite™ and dialyzed against ethanol for 2 d. After filtering, the solvent was

evaporated under pressure and product was dried in a vacuum oven at 45°C overnight and then

collected.

Pullulan Suberate Acetate Yield: (56%). 1H NMR (CDCl3): 1.8-2.2 (COCH3 of acetate), 1.25

(COCH2CH2CH2CH2CH2CH2CO of suberate), 1.54 (COCH2CH2CH2CH2CH2CH2CO of suberate), 2.27

(COCH2CH2CH2CH2CH2CH2CO of suberate), 3.4-5.5 (pullulan backbone).

3.4 Results and Discussion

3.4.1 Synthesis of pullulan ω-carboxyalkanoates

The approach used in the synthesis of these pullulan derivatives varied from the

previously reported synthesis of cellulose ω-carboxyalkanoates using adipic anhydride. As stated

previously, reaction with adipic anhydride resulted in cross-linked cellulose esters and

insolubility. Additionally, because suberate or sebacate anhydrides are not sufficiently stable to

isolate and use, another method had to be implemented. Therefore, adipic, suberic, and sebacic

acids were the starting materials, and were mono-protected as benzyl esters to avoid the

possibility of crosslinking. In previous publications, monobenzyl esters of diacids, activated at the

other carbonyl as acid chlorides, were reacted with preformed cellulose esters, since these

cellulose esters have far superior organic solubility to cellulose and thereby permit a wide choice

of reaction solvents for the acylation with, e.g., monobenzyl adipoyl chloride.

Due to the commercial unavailability of pullulan esters, synthesis of pullulan ω-

carboxyalkanoates began with the naturally occurring polysaccharide. Initially attempts were

made to react monobenzyl esters of the diacids, after conversion to their monoacid chlorides,

with pullulan alkanoates that we synthesized (e.g. pullulan acetate). We observed that reaction

51

with the diacid derivatives did afford pullulan alkanoate -carboxyalkanoates as anticipated, but

often with unsatisfactory DS (-carboxyalkanoate) (see Appendix). In order to increase the DS of

-carboxyalkanoate substituents, the less reactive long chain acid chlorides were added first,

followed by the addition of acid anhydrides (Figure 3.1). In this way they could react with the

most reactive hydroxyl groups (i.e., the less-hindered primary hydroxyl at C-6) before the more

reactive carboxylic anhydride entity could compete. This order of addition successfully afforded

pullulan derivatives with high DS values of -carboxyalkanoate esters in each of the adipate,

suberate, and sebacate series (Table 3.1). Simply by adjusting stoichiometry, we could also

synthesize mixed esters with lower DS -carboxyalkanoates, in order to provide a wide range of

derivatives with different levels of hydrophobicity and carboxyl content.

Pullulan Derivative Abr. DS ω-carboxy DS alkanoate Hydroxyl Content

Pullulan Acetate Adipate PullAcAd

1.13 1.72 0.15 Pullulan Acetate Adipate * 0.51 1.82 0.67 Pullulan Adipate Propionate

PullAdPr 1.05 1.52 0.43

Pullulan Adipate Propionate* 0.50 1.54 0.96 Pullulan Adipate Butyrate

PullAdBut 1.08 1.16 0.76

Pullulan Adipate Butyrate* 0.47 1.44 1.09 Pullulan Acetate Suberate

PullAcSub 0.90 1.83 0.27

Pullulan Acetate Suberate * 0.61 2.03 0.36 Pullulan Propionate Suberate

PullPrSub 1.00 1.47 0.53

Pullulan Propionate Suberate * 0.68 1.50 0.82 Pullulan Butyrate Suberate

PullButSub 1.03 1.51 0.46

Pullulan Butyrate Suberate * 0.68 1.37 0.95 Pullulan Acetate Sebacate

PullAcSeb 0.88 1.93 0.19

Pullulan Acetate Sebacate * 0.51 1.95 0.54 Pullulan Propionate Sebacate

PullPrSeb 0.99 1.71 0.30

Pullulan Propionate Sebacate * 0.51 1.62 0.87 Pullulan Butyrate Sebacate

PullButSeb 0.88 1.38 0.74

Pullulan Butyrate Sebacate * 0.67 1.32 1.01

*Derivatives were made using 2 eq. of Acid Chloride; -carboxyalkanoate = (Adipate/Suberate/Sebacate); alkanoate = (Acetate/Propionate/Butyrate)

Table 3.1: Comparison of DS ω-carboxy, DS alkanoate, and hydroxyl group content of each pullulan derivative.

52

The monobenzyl pullulan -carboxyalkanoate ester derivatives were confirmed by 1H

NMR and 13C NMR to be completely incorporated into the pullulan backbone with no evidence

of cross-linked product (Figure 3.2). Confirmation by 1H NMR analysis of monobenzyl pullulan

propionate suberate showed a singlet for the phenyl hydrogens downfield at 7.32 ppm and

benzylic hydrogen peak further upfield at 5.2 ppm relative to the solvent. Protons along the

suberoyl chain were seen upfield at 1.26, 1.55 and 2.26 ppm. The pullulan backbone occupied

the region of 3.25 – 6.0 ppm and protons from the CH3 of the propyl group shifted upfield at 1.0

ppm. 13C NMR further confirmed the structure showing phenyl and benzyl carbon shifts

appearing at 127 and 65 ppm respectively. Carbonyl carbons from both the propionate and

suberoyl groups shifted downfield to 173 ppm and the methyl carbon of the propionate group

shifted upfield to 9 ppm. The pullulan backbone occupied the region 57 – 73 ppm and the

anomeric carbon shifted downfield to 95 ppm.

ATR-FTIR spectroscopy analysis of monobenzyl pullulan propionate suberate showed

strong absorption in the region of 3400 cm-1, which is typical for O-H stretch region for alcohols

(Figure 3.3). Alkanoate functionality was confirmed by carbonyl stretching seen in the region of

1700 cm-1 followed by ester and alcohol stretching in the region of 1200 cm-1 and 1000 cm-1.

Benzyl group functionality was also seen in the region of 700 cm-1 which is typical for aromatic

compounds.

53

Figure 3.2: (a) 1H NMR and (b) 13C NMR of Monobenzyl Pullulan Suberate Propionate in d6-

DMSO and CDCl3 respectively.

9 13 12

8,11 14

16

1-6

9

13

12 11 14

1

16

15

7,10 2-6 8

54

Figure 3.3: ATR-FTIR spectra of monobenzyl pullulan suberate propionate

Hydrogenolysis of these monobenzyl pullulan esters using a heterogeneous method,

Pd(OH)2/C in THF under pressure for 2 days, only partially removed the benzyl groups, unlike in

the case of cellulose -carboxyalkanoates where complete hydrogenolysis was observed under

otherwise equivalent conditions. It is possible that the random coil structure of pullulan makes

interaction with the insoluble catalyst surface more difficult. To rectify this issue, hydrogenolysis

was carried out using both hydrogen and triethylsilane, which is a hydrogen donor in solution

and has been reported to be efficient in the deprotection of benzyl esters and other bulky groups

31. This modified process afforded complete hydrogenolysis of benzyl groups and showed no

residual presence of phenyl or benzyl resonances in either 1H NMR or 13C NMR spectra (Figure

3.3). 1H NMR spectra of pullulan propionate suberate showed no phenyl and benzylic hydrogen

in the regions of 7.23 and 5.2 ppm respectively. Additionally, 13C NMR showed the absence of

phenyl and benzyl carbons at 127 and 65 ppm respectively.

5001000150020002500300035004000

Ab

sorb

ance

Wavenumber (cm-1)

O-H stretch

C-H stretch

C=O stretch

C-O stretch Aromatic

Ring

55

Figure 3.4: (a) 1H NMR and (b) 13C NMR spectra of pullulan propionate suberate in d6-DMSO

showing the absence of phenyl and benzyl peaks. 10* denotes the carbonyl carbon adjacent to

the ω-carboxy group.

1-6

9 13

3 12

11

8

9

No phenyl peak

13 12

11

8

2-6

No benzylic peak

7,10

10*

1

No phenyl peak No benzylic peak

56

Further confirmation of the structure of the ω-carboxyalkanoate was obtained by ATR-

FTIR spectroscopy (Figure 3.4). The pullulan suberate alkanoate (PullAcSub0.61) absorbed

strongly in the region from 3600 to 3100 cm-1, encompassing the expected region for O-H stretch

of the alcohols and carboxylic acids, which is confirmation that unsubstituted OH groups and

carboxyl functionality are present as expected in these polymers. A sharp multiplet C-H stretch

was seen at 2900 cm-1, which is typical for alkanes. Ester and acid carbonyl functionality was also

observed based on the large C=O stretch seen at 1750 cm-1. The presence of esters was also

confirmed by C-O stretching seen at 1250 cm-1 and 1000 cm-1. Since these polymers differ most

significantly only in -carboxyalkanoate chain length and DS, it is to be expected that the spectra

look very similar to one another. Based on the evidence of 13C and 1H NMR spectra, and FTIR

data, this methodology was successful for synthesis of pullulan ω-carboxyalkanoate mixed esters.

Figure 3.5: Comparison of the ATR-FTIR spectra of pullulan suberate

acetate/propionate/sebacate

57

3.4.2 Thermal properties

It is very important that polymers chosen as ASD candidates have high glass transition

temperatures (Tg). This is to ensure that the drug/polymer matrix maintains Tg above any possible

ambient temperature during transport and storage before it reaches patients. Polymers that

exhibit low Tg values allow for molecular mobility of the chains and therefore migration of the

drug particles in the matrix, which may lead to recrystallization22. Ideally, the Tg should be well

above ambient temperature due to the possibility of a plasticization effect caused by either the

drug or high humidity. Similarly, polymers that have low decomposition temperatures (Td’s) are

also less suitable for ASD application, since melt extrusion is a favored method for production of

drug/polymer ASDs by many producers.

DSC analysis of these pullulan derivatives show that they display overall lower values of

Tg than for analogous derivatives of the rigid rod polysaccharide cellulose (Table 3.2).

PullAcSub0.9 had the highest Tg at 93oC. It is clear that there is a strong relationship between

chain length of substituent, substituent DS, and glass transition temperature. Clearly the

substituents are acting as internal plasticizers; so much so that the pullulan ester with the longest

chain substituents PullButSeb0.88 had a glass temperature well below ambient. While increasing

alkanoate chain length consistently led to a decrease in Tg across all pullulan esters synthesized,

the effect of ω-carboxyalkanoate DS was less consistent (Figure 3.6). For the pullulan adipates,

higher DS(ω-carboxyalkanoate) consistently led to decreasing Tg. For the pullulan suberates, a

small increase in Tg was observed with increasing DS(ω-carboxyalkanoate), and for the sebacates

too few samples showed a clear glass transition for a clear trend to be discernable.

Upon further investigation of the pullulan sebacate derivatives, the thermal transitions

are far lower than those of the shorter chain suberate derivatives. The highest Tg was observed

from PullAcSeb0.51 at 38.54 oC. The only other observable Tg came from PullAcSeb0.88 at -43.91

oC and PullPrSeb0.51 at -49.13 oC. As seen with the suberate derivatives an increase in

hydrophobicity did lead to a decrease in Tg and though no transitions were seen for the rest of

the sebacate derivatives, the DSC thermograms did indicate some crystallinity due to the

presence of a melting endotherm (Figure 3.6). The sebacoyl side chains, though being long and

58

hydrophobic, still find a way to orient themselves to form crystalline domains, which brings up

the possibility of substitution regularity along the pullulan backbone.

PullAcAd0.51 and PullAdPr0.5 exhibited high Tg’s at 86 oC and 88 oC respectively. A small

decrease in thermal stability was observed with an increase in alkanoate chain length. More

surprisingly, both the adipate butyrate derivatives showed no visible Tg, but like some of the

sebacate derivatives, a melting endotherm was observed which is confirmation of crystallinity.

Sample Tg (oC) Td (oC) SP (MPA1/2)

a b a b A b

Pullulan Adipate Acetate 86.23 47.24 371.58 370.41 23.07 22.44

Pullulan Adipate Propionate 88.22 35.69 359.23 342.54 23.32 22.59

Pullulan Adipate Butyrate - - 313.29 325.87 23.18 23.10

Pullulan Suberate Acetate 81.10 93.32 359.19 379.91 22.07 22.06

Pullulan Suberate Propionate 79.43 85.62 358.61 377.84 22.75 22.30

Pullulan Suberate Butyrate 68.74 80.63 364.32 376.98 22.71 21.89

Pullulan Sebacate Acetate 38.54 -43.91 358.72 335.85 22.16 21.54

Pullulan Sebacate Propionate -49.13 - 355.17 320.63 22.49 21.48

Pullulan Sebacate Butyrate - - 353.29 317.29 22.54 22.02

(-): Samples exhibited no observable Tg ; SP = Solubility Parameter

a: Prepared using 2 equiv. of acid chloride

b: Prepared using 3 equiv. of acid chloride

Table 3.2: TGA and DSC values for pullulan alkanoate ω-carboxyalkanoates and their solubility

parameters

59

Figure 3.6: DSC thermograms of pullulan suberate derivatives showing the Tg as well as PullPrSeb0.99 showing endotherm.

TGA analysis of the decomposition temperatures showed all the derivatives exhibiting Td’s

above 300 oC (Table 3.2). PullAcSub0.9 had the highest at 380 oC and PullButAd0.47 had the

lowest at 313 oC. Higher stability was seen as ω-carboxy content decreased for the adipoyl and

sebacoyl series, but suberoyl derivatives showed a slight increase a carboxy content increased.

Further analysis showed than the increase in alkanoate chain length did lead to a slight decrease

in Td, but remained relatively high (Figure 3.7). Higher stability overall did come from the

suberate derivatives, and this can be due to the suberate chain to possessing the right balance

of hydrophobicity and hydrophilicity to allow polymer chains to orient more efficiently allowing

for higher thermal properties.

60

Figure 3.7: TGA thermograms of pullulan suberate derivatives 3.4.3 Solubility

In order for these pullulan derivatives to be useful in a variety of applications, it is

important that they display adequate solubility for processing. All of the pullulan ω-

carboxyalkanoates exhibited complete solubility in polar aprotic solvents such as THF and DMSO

(Table 3.3). Similarly, bulk insolubility was observed in all derivatives in water and hexanes.

Derivatives made with lower ω-carboxyalkanoyl content were completely soluble in acetone as

were the higher DSω-carboxy suberate derivatives excluding suberate acetate, which showed partial

solubility. Solubility in polar protic solvents was observed as well. Ethanol served as a good

solvent for lower DS(ω-carboxyalkanoyl) derivatives excluding PullAcSub0.61 and PullAcAd0.51;

it is well known that polysaccharide esters increase in solubility going from acetate to propionate

to butyrate. Further increase in DS(ω-carboxyalkanoyl) however, produced insolubility in

ethanol.

Temperature (oC)

Wei

ght

Per

cen

tage

x

Td: 358.61oC

x x

Td: 359.61oC

Td: 364.32oC

50% wt. loss

--- PullButSub0.68

--- PullPrSub0.68

--- PullAcSub0.61

61

Sample H2O THF EtOH CHCl3 DMSO DMK Hex

a b a b a b a b a b a b a b

Pullulan Adipate Acetate x x o o x x x x o o o p x X

Pullulan Adipate Propionate x x o o p o x x o o o p x x

Pullulan Adipate Butyrate x x o o o x x x o o o p x x

Pullulan Suberate Acetate x x o o x o x x o o o x x x

Pullulan Suberate Propionate x x o o o x x x o o o x x x

Pullulan Suberate Butyrate x x o o o x x x o o o x x x

Pullulan Sebacate Acetate x x o o o p x x o o o p x x

Pullulan Sebacate Propionate x x o o o x x x o o o o x x

Pullulan Sebacate Butyrate x x o o o x x x o o o o x x

H2O water, THF tetrahydrofuran, EtOH ethanol, CHCl3 chloroform DMSO Dimethylsulfoxide DMK acetone Hex hexanes o = Soluble p = Partially Soluble x = Insoluble a: Prepared using 2 equiv. of acid chloride

b: Prepared using 3 equiv. of acid chloride

Table 3.3: Solubility of pullulan alkanoate ω-carboxyalkanoates in various solvents

3.4.4 Solubility parameters

As described earlier, the solubility parameter (SP) is a calculated value that indicates the

relative polymer hydrophobicity, and is based on group contributions. In this work, we used the

methods of Fedors for the SP calculation. Previous SP evaluation of cellulose ω-

carboxyalkanoates reported that samples that had calculated SP values within the range of 20.56

– 22.62 MPa1/2, were effective in inhibiting the crystal growth of ritonavir; SP was a significant

predictor of a polymer’s ability to function as an ASD polymer22.

Table 3.2 shows the solubility parameter calculations for all the pullulan ω-

carboxyalkanoates based on measured DS values of ω-carboxyalkanoyl and alkanoyl, converted

into group contributions. It should be noted however, that using this group contribution method,

cellulose and pullulan are indistinguishable; the methodology cannot account for the three

dimensional shape differences between rigid rod cellulose and random coil pullulan. The highest

solubility parameter was calculated to be 23.32 MPa1/2 and was displayed by PullAcAd (DSω-carboxy

= 0.50, DSalkanoate = 1.54). The lowest was calculated to be 21.48 MPa1/2 and was displayed by

PullPrSeb (DSω-carboxy = 0.99, DSalkanoate = 1.71). Based on these calculations, all synthesized

pullulan ω-carboxyalkanoate alkanoates fall within the promising SP range, meriting further

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testing as ASD candidates. This will of course need to be confirmed, and the effects of three

dimensional structure ascertained, by evaluation in ASDs.

3.5. Conclusion

Novel pullulan ω-carboxyalkanoates were successfully synthesized by a methodology that

takes advantage of the differing reactivity of pullulan hydroxyls, with a sequence that permits

high DS(-carboxyalkanoate) if desired, and gives good control over DS of both substituents

simply by changing stoichiometry. Likewise, issues with slow heterogeneous hydrogenolysis were

overcome by using a soluble hydrogen source, to yield a fully deprotected product. Derivatives

displayed adequate solubility in polar protic and polar aprotic solvents with the derivatives with

lower ω-carboxyalkanoyl content showing complete solubility in acetone, and in many cases, in

ethanol (as acetone and ethanol are preferred solvents for spray drying of ASDs, due to their low

toxicities, this is promising). Pullulan suberates displayed the most promising thermal properties,

due to a favorable balance of hydrophobicity and hydrophilicity. PullAcSub0.9 exhibited the

highest Tg at 93 oC and although PullSebProp0.99 did not show an observable Tg, the presence of

a melting endotherm confirms some side chain crystallinity. Melting endotherms were also

observed for pullulan sebacate butyrate and the pullulan adipate butyrate derivatives. All of the

derivatives showed high thermal stability and recorded Td’s above 300 oC. The pullulan suberate

series, PullAcAd0.51 and PullAdPr0.5 could be the best choices for continued study as potential

ASD polymers. Their high Tg values as well as their solubility parameters fall in the range to be

acceptable as good candidates. Since we don’t yet know how the three dimensional structure of

these pullulan alkanoate w-carboxyalkanoates will impact their miscibility with drugs and their

ability to stabilize drugs against crystallization, and since all displayed SP values are in the range

that has previously been shown to be effective for ASD, they are all worthy of further study for

ASD and other drug delivery applications.

63

3.6 Acknowledgements

This project was possible due the kind donation of pullulan from the Hayashibara

Company. We thank Laura Mosquera-Giraldo (Purdue University) for calculating the solubility

parameters. We would like to thank the National Science Foundation for their financial support

through grant DMR-1308276. We would also like to the Macromolecules and Interfaces Institute

and Institute for Critical Technology and Applied Sciences for their facilities and educational

support.

3.7 References

1. Silman, R. W.; Bryan, W. L.; Leathers, T. D., A comparison of polyssacharides from strains of Aureobasidium pullulans. FEMS Microbiology Letters 1990, 71 (1–2), 65-70.

2. West, T. P.; Reed-Hamer, B., Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans. FEMS Microbiology Letters 1993, 113 (3), 345-349.


4. Kaplan, D. L.; Mayer, J.; Lombardi, S.; Wiley, B.; Arcidiacono, S., Biodegradable polymers for material applications chitosan and pullulan. Abstracts of Papers American Chemical Society 1989, 197, 53.

5. Yamaoka, T. Y., T. & Ikada, Y., Body distribution profile of polysaccharides after intravenous administration. Drug Delivery 1993, 1 (1), 8.

6. Prajapati, V. D.; Jani, G. K.; Khanda, S. M., Pullulan: An exopolysaccharide and its various applications. Carbohydr. Polym. 2013, 95 (1), 540-549.

64

7. Xiao, Q.; Tong, Q.; Lim, L.-T., Drying process of pullulan edible films forming solutions studied by ATR-FTIR with two-dimensional correlation spectroscopy. Food Chemistry 2014, 150 (0), 267-273.

8. Kristo, E.; Biliaderis, C. G.; Zampraka, A., Water vapour barrier and tensile properties of composite caseinate-pullulan films: Biopolymer composition effects and impact of beeswax lamination. Food Chemistry 2007, 101 (2), 753-764.

9. Alonzo, D. E.; Zhang, G. G.; Zhou, D.; Gao, Y.; Taylor, L. S., Understanding the behavior of amorphous pharmaceutical systems during dissolution. Pharmaceutical research 2010, 27 (4), 608-18.


11. Wong, V. W.; Rustad, K. C.; Galvez, M. G.; Neofyotou, E.; Glotzbach, J. P.; Januszyk, M.; Major, M. R.; Sorkin, M.; Longaker, M. T.; Rajadas, J.; Gurtner, G. C., Engineered Pullulan-Collagen Composite Dermal Hydrogels Improve Early Cutaneous Wound Healing. Tissue Eng. Part A 2011, 17 (5-6), 631-644.

12. Gupta, M.; Gupta, A. K., Hydrogel pullulan nanoparticles encapsulating pBUDLacZ plasmid as an efficient gene delivery carrier. Journal of Controlled Release 2004, 99 (1), 157-166.

13. Dulong, V.; Cerf, D. L.; Picton, L.; Muller, G., Carboxymethylpullulan hydrogels with a ionic and/or amphiphilic behavior: Swelling properties and entrapment of cationic and/or hydrophobic molecules. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2006, 274 (1–3), 163-169.

14. Jung, S.-W.; Jeong, Y.-I.; Kim, S.-H., Characterization of hydrophobized pullulan with various hydrophobicities. International Journal of Pharmaceutics 2003, 254 (2), 109-121.


16. Martina, B.; Katerina, K.; Miloslava, R.; Jan, G.; Ruta, M., Oxycellulose: Significant Characteristics in Relation to Its Pharmaceutical and Medical Applications. Adv. Polym. Technol. 2009, 28 (3), 199-208.

17. Zhang, R.; Tang, M.; Bowyer, A.; Eisenthal, R.; Hubble, J., A novel pH- and ionic-strength-sensitive carboxy methyl dextran hydrogel. Biomaterials 2005, 26 (22), 4677-4683.

65

18. Nam, K. W.; Watanabe, J.; Ishihara, K., pH-modulated release of insulin entrapped in a spontaneously formed hydrogel system composed of two water-soluble phospholipid polymers. J. Biomater. Sci.-Polym. Ed. 2002, 13 (11), 1259-1269.

19. Konno, H.; Handa, T.; Alonzo, D. E.; Taylor, L. S., Effect of polymer type on the dissolution profile of amorphous solid dispersions containing felodipine. European Journal of Pharmaceutics and Biopharmaceutics 2008, 70 (2), 493-499.


21. Kar, N.; Liu, H.; Edgar, K. J., Synthesis of Cellulose Adipate Derivatives. Biomacromolecules 2011, 12 (4), 10.



24. Teramoto, N.; Shibata, M., Synthesis and properties of pullulan acetate. Thermal properties, biodegradability, and a semi-clear gel formation in organic solvents. Carbohydr. Polym. 2006, 63 (4), 476-481.

25. Zhang, H. Z.; Zhang, Q. Q., Preparation of Folate Targeted Pullulan Acetate Nanoparticles and Cell Uptake in vitro. Chem. J. Chin. Univ.-Chin. 2009, 30 (6), 1146-1151.


27. Li, B.; Konecke, S.; Harich, K.; Wegiel, L.; Taylor, L. S.; Edgar, K. J., Solid dispersion of quercetin in cellulose derivative matrices influences both solubility and stability. Carbohydr Polym 2013, 92 (2), 2033-40.

28. Chen, Y. J.; Liu, C. Y.; Chen, Z.; Su, C.; Hageman, M.; Hussain, M.; Haskell, R.; Stefanski, K.; Qian, F., Drug-Polymer-Water Interaction and Its Implication for the Dissolution Performance of Amorphous Solid Dispersions. Mol. Pharm. 2015, 12 (2), 576-589.

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29. Fishman, M. L.; Damert, W. C.; Phillips, J. G.; Barford, R. A., Evaluation of root-mean-square radius of gyration as a parameter for universal calibration of polysaccharides. Carbohydr. Res. 1987, 160, 215-225.

30. Fedors, R. F., Method for estimating both solubility parameters and molar volumes of liquids. Polymer Engineering and Science 1974, 14 (2), 147-154.

31. Mandal, P. K.; McMurray, J. S., Pd-C-induced catalytic transfer hydrogenation with triethylsilane. J. Org. Chem. 2007, 72 (17), 6599-6601.

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CHAPTER 4: Summary and Future Work

4.1 Summary

Successful synthesis of pullulan ω-carboxyalkanoates has herein been reported. Utilizing

methods adapted from Liu et.al.1 for the synthesis of cellulose ω-carboxyalkanoates, a novel set

of new pullulan derivatives with promising properties have been prepared. High DS of both ω-

carboxy and alkanoate groups could be achieved through the reaction of pullulan with the less

reactive monobenzyl chloride moieties first, followed by reaction with alkanoyl chlorides.

Successful incorporation into the pullulan backbone with no evidence of cross-linked product was

confirmed by 1H NMR and 13C NMR. Deprotection of these benzyl groups to allow for carboxy

functionality was successfully achieved through hydrogenolysis utilizing Pd(OH)2/C as a catalyst

and triethylsilane as a proton donor in solution to afford a deprotected product. This was also

confirmed through 1H NMR and 13C NMR as well as FTIR spectra.

Products with a wide range of solubility parameters were obtained, primarily by varying

the hydrocarbon chain length as well as by varying the DS of the carboxyl side chains. It should

be noted however, that these calculations do not distinguish pullulan from cellulose. Solubility

parameters only consider group contribution, not three dimensional structure (e.g. linear

cellulose vs. random coil pullulan). Given the greater solubility of natural pullulan than cellulose,

it is not surprising that solubilities of otherwise similar pullulan derivatives would be increased

vs. those of cellulose as well. By designing such that there were residual unsubstituted hydroxyl

groups on the pullulan backbone, thereby enhancing water solubility, we were able to obtain

amphiphilic polymers with desired solubility characteristics.

Thermal analysis of these polymers showed that they all have high decomposition

temperatures above 300oC. Similarly, high glass transition temperatures were observed for all of

the pullulan suberate series as well as PullAcAd0.51 and PullAdPr0.51. Thermal properties were

affected by chain length and DS, adipoyl and sebacoyl derivatives with higher ω-carboxy content

had slightly decreased decomposition temperatures. However, increase in alkanoate chain length

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did lead to a decrease in Td’s across all the series. Lower carboxy content and shorter alkanoate

chain length however did show an increase in Tg’s.

Suberoyl containing polymers as well as PullAcAd0.51 and PullAdPr0.5 show the most

promise as good candidates for ASD formulations. They have high glass transition temperatures

and solubility parameters within a range similar to those of many hydrophobic drugs, and similar

to polymers that have previously shown promise in ASD. However due to a lack a knowledge on

how the three dimensional structure of these derivative will affect drug miscibility, all should still

be considered.

4.2 Future Work

4.2.1 Synthesis of pullulan ω-carboxyalkanoates by olefin cross-metathesis (OCM)

Recently, data has been published on the successful use of olefin cross metathesis on

cellulose to provide a large range of different functionalities2-4. Specifically, Meng et. al. were

able to successfully synthesize cellulose ω-carboxyalkanoates using OCM and Hoveyda-Grubbs

second generation catalyst (Figure 4.1). The products were soluble in various organic solvents

and showed high glass transition temperatures; however, in these derivatives the double bond

was preserved. It was also reported that crosslinking was observed after being in storage for an

extended period and was attributed to Michael addition of the α,β-unsaturated carboxylic acid

groups. Meng did show however, that complete hydrogenation of these double bonds made via

cross metathesis on cellulose derivatives was possible, and that the resulting saturated

derivatives had good stability.

69

Figure 4.1: Schematics of synthesis of cellulose ω-carboxyalkanoates via olefin cross-metathesis adapted from Meng et. al.

Due to the ability of pullulan to form random coils in solution, the use of triethylsilane

(TES) was implemented to help in the hydrogenolysis process. Due to the addition of this reducing

agent, filtration and purification of the pullulan derivatives were very time consuming. A

reasonable way going forward in the synthesis of these compounds would be through olefin

cross-metathesis. Through this method, the degree of substitution of the ω-carboxy substituents

could be precisely controlled and the issue of bulky substituents being inaccessible would be

greatly reduced. The use of olefin cross-metathesis for the synthesis of pullulan ω-

carboxyalkanoates would be able to provide a more efficient and less time-consuming means for

synthesis by eliminating the need for deprotection of the benzyl group. Additionally, Michael or

thio-Michael addition through the double bond will allow for increased terminal functionality,

the ability to vary chain length, and to introduce branched groups.

4.2.2 Amorphous Solid Dispersions

As discussed in Chapters 2 and 3, ASDs work by blending a hydrophobic crystalline drug

into a polymer matrix to retard crystallinity and increase apparent solubility and bioavailability

through the enterocytes of the gastrointestinal (GI) tract. Additionally, these polymers should

exhibit a high glass transition temperature to prevent recrystallization and a pH-sensitive

mechanism for release. This ensures that during the acidic environment of the stomach, the

matrix remains intact, but when introduced to the neutral pH of the GI tract, swelling occurs and

the drug is released to be absorbed (Figure 4.2).

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Figure 4.2: Schematic of how an Amorphous Solid Dispersion works One of the main goals for the synthesis of these pullulan ω-carboxyalkanoates was to try

and improve upon their cellulosic counterparts which showed very good promise as ASD polymer

candidates. Good candidates that exhibited useful ASD properties were suberoyl-containing

polymers as well as pullulan adipate acetate (DSω-carboxy = 0.51) and pullulan adipate propionate

(DSω-carboxy = 0.50). Previous literature has shown that the cellulose ω-carboxyalkanoates are able

to retard the recrystallization of the anti-HIV drug ritonavir and the antibacterial clarithromycin5-

6. These polymers exhibit high glass transitions, usually at least 50oC above ambient temperature,

but are limited due to their water insolubility. A small amount of polymer dissolved in water is

essential in order to help prevent recrystallization of the drug after it is released in the GI tract.

Not all the pullulan derivatives exhibit a high enough Tg for this application, but there are a few

that do. Moving forward, these derivatives should first be tested for their ability to retard the

recrystallization of hydrophobic drugs. Next would be dissolution studies as well as analysis of

the ASD matrix to see if it is stable and if the drug can be dispersed in its matrix. Through the

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dissolution studies, the effectiveness of the polymer matrix in various conditions simulating the

GI tract can be obtained and conclusions can be drawn on the structure/ASD property

relationships, and the polymers’ potential in ASD formulations.

4.2.3 Other Uses

Due to pullulan’s unique ability to be cleared from the body and not invoke an immune

response, the use of these derivatives in other modes of drug delivery, primarily intravenous

admistration can be studied. Toxicity tests on live cells should be the first course of action given

to see how the cells respond. Though natural pullulan may be non-toxic, it cannot be assumed

that these derivatives would be also. An analysis of their in vivo biodegradation, as well as their

ability to invoke immune responses would help indicate whether these derivatives maintained

toxicity properties similar to the natural polymer.

Even though not all the pullulan ω-carboxyalkanoates showed sufficiently high glass

transition temperatures for use as ASD matrices, they may be valuable for other applications.

Due to the hydrophilicilty of the carboxy and hyroxyl groups and the hydrophobicity from the

backbone and alkanoate groups, these pullulan derivatives exhibit amphiphillic characteristics.

The applications that may be enabled due to this amphiphilic property should be explored.

Dynamic Light-Scattering (DLS) can be used to test the ability of these derivatives to form

micelles. Futher analysis of DLS data could be used to measure micelle size and surface tension

measurements to find the critical micellece concentrations (CMCs). Previous work done on the

synthesis of ampiphilic 6-carboxypullulan 2, 3-O-ethers afforded derivatives that exhibited very

low critical micelle concentrations (CMCs)7. This supports the hypothesis that pullulan derivatives

may be attractive for surfactant and other applications that benefit from amphiphilicity.

Exploration of pH dependence on the solubility and micellization properties would

provide better insight on how 3D structure changes with respect to pH. Additonally, changes in

the amphilicity of the polymer could be studied. Correlations between observed solubility and

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calculated solubility parameter would also be interesting to explore to see if the solubility

parameter calculation has any predictive inferences on the solubility of these derivatives.

Additionally, correlation on the foaming ability of some of these derivtives versus their solubility

parameter calculation would be of great interest as well.

4.3 References


2. Dong, Y.; Edgar, K. J., Imparting functional variety to cellulose ethers via olefin cross-metathesis. Polymer Chemistry 2015, 6 (20), 3816-3827.

3. Meng, X.; Matson, J. B.; Edgar, K. J., Olefin cross-metathesis, a mild, modular approach to functionalized cellulose esters. Polymer Chemistry 2014, 5 (24), 7021-7033.

4. Meng, X.; Matson, J. B.; Edgar, K. J., Olefin Cross-Metathesis as a Source of Polysaccharide Derivatives: Cellulose omega-Carboxyalkanoates. Biomacromolecules 2014, 15 (1), 177-187.


6. Pereira, J. M.; Mejia-Ariza, R.; Ilevbare, G. A.; McGettigan, H. E.; Sriranganathan, N.; Taylor, L. S.; Davis, R. M.; Edgar, K. J., Interplay of Degradation, Dissolution and Stabilization of Clarithromycin and Its Amorphous Solid Dispersions. Mol. Pharm. 2013, 10 (12), 4640-4653.


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APPENDIX

Chapter 3: Synthesis of pullulan ω-carboxyalkanoates for drug nanodispersions

Figure A3.1: 1H NMR spectra of monobenzyl pullulan acetate/propionate/butyrate

Pullulan Acetate

DS: ≈2.0

Pullulan Propionate

DS: ≈2.0

Pullulan Butyrate

DS: ≈1.67

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Figure A3.2: 1H NMR spectra of monobenzyl pullulan acetate adipate/suberate/sebacate made starting

from pullulan acetate DS 2.0

PullAcAd

DS Ad: ≈0.71

PullAcSub

DS Ad: ≈0.18

PullAcSeb

DS Seb: ≈0.24

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Figure A3.2: 1H NMR spectra of monobenzyl pullulan adipate acetate/suberate/sebacate made starting

from natural pullulan

Figure A3.3: 1H NMR spectra of monobenzyl pullulan suberate acetate/suberate/sebacate made starting


PullAdBut

DS Ad: ≈1.18

PullAdPr

DS Ad: ≈1.05

PullAcAd

DS Ad: ≈1.13

PullButSub

DS Ad: ≈1.03

PullPrSub

DS Ad: ≈1.0

PullAcSub

DS Ad: ≈0.99

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Figure A3.3: 1H NMR spectra of monobenzyl pullulan sebacate acetate/suberate/sebacate made starting


Figure A3.4: 1H NMR spectra of monobenzyl pullulan suberate acetate (a) before and (b) after

hydrogenolysis for 2 d in Parr Reactor with Pd(OH)2 catalyst

PullButSeb

DS Ad: ≈0.88

PullPrSeb

DS Ad: ≈0.99

PullAcSeb

DS Ad: ≈0.88

(a) PullAcSub

DS Sub: ≈0.90

(b) PullAcSub

DS Ad: ≈0.73

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Figure A3.5: 1H NMR spectra of pullulan acetate adipate/suberate/ sebacate after hydrogenolysis with

TES for 2 d.

PullAcAd

PullAcSub

PullAcSeb

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