Pullulan Ω-Carboxyalkanoates for Drug Nanodispersions Jameison T

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

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.

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.

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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!!!

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

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

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

1 13 Figure 3.2: (a) H NMR and (b) C 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

1 13 Figure 3.4: (a) H NMR and (b) C 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

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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

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

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.

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.

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-(14) 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-(16) 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-(14) and α-(16) linkages in the ratio of 2:114. Therefore, pullulan is depicted as a linear polysaccharide having a maltotriosoyl [α-D-Glcp-(14)-α-D-Glcp-(14) )-α-D-Glcp-(14)] repeat unit connected by α-(16) linkages. The trisaccharide repeat unit is represented by the nomenclature [4)-α-D-Glcp-(16)-α-D- Glcp-(14)-α-D-Glcp-(1] (Figure 2.1).

Pullulan however, can also be described as a polymer of panose [α-D-Glcp-(16)-α-D- Glcp-(14)-α-D-Glcp] and isopanose [α-D-Glcp-(14)-α-D-Glcp-(16)- 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-(14)-Glcp-α- (14)- Glcp-α-(14)-Glcp-α-(16)-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.

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

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.

Fructose 1,6 Polysaccharides Monosaccharides Fructose bisphosphate Amylases Isomerases 2x ATP Phosphorylation

Fructose 6 Glucose Glucose 6 phosphate phosphate Phosphorylation Isomerase n

α-phosphoglucose mutase

Glucose 1 phosphate

UDPG-pyrophosphorylase

UDP-Glc

UDP LPh

LPh-Glc

UDPG

UDP

LPh-Glc-(6←1)-Glc Isomaltosyl

LPh-Glc-(6←1)-Glc-(4←1)-Glc Isopanosyl

glucosyltransferase

Pullulan

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

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-(13) units (Table 2.1).

Microbes Used Ratio of Variations of Structure References α-(14): α-(16) linkages Aureobasidium 2; 1.5 Contains up to 7% 11, 16 pullulans* maltotetrose units; up to 6% of α-(13) linkages Tremella mesenterica 2 25 Cytaria harioti 2.4 3-7 % α-(13) linkages 26 Cytaria darwinii 2 27 Cryphonectria parasitica 2.3-2.4 Up to 89% maltotetrose 28 units 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

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- (16) 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

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 THF Chloroform Toluene Acetate 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

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

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 Neuronal gene delivery 54 anioplexes Carboxymethyl pullulan Thermoassociative particles 55-56 Hydrogels Pullulan Sulfates Transmucosal protein delivery 57-58 Anticoagulants Phosphorylated pullulan Implant surfaces 59 Succinylated pullulan Thermosensitive electrostatic complexes 60-61 copolymers Pullulan acetate Nanoparticles 62-63 Cholesteryl pullulan pH sensitive gels 64

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

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

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

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 PullAc PropylPull PropylPull ButylPull ButylPull DS: 1.0 DS: 2.4 DS: 1.02 DS: 2.45 DS: 1.28 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

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.

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.

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.

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

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

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.

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

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

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

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

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

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.

2.7 References