Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, Division of Biochemistry, 1966 / In title on t.p., "[epsilon]" appears as the lower-case Greek letter. "September, 1966." / Includes bibliographical references (leaves 157-165). / by John Mahlon Keller. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biology, Division of Biochemistry

Cellulose ethers are valuable matrices for drug-delivery systems (DDS), namely amorphous solid dispersions (ASD). ASD are efficient vehicles that can solubilize and stabilize poorly soluble drugs by increasing the time that it takes for drugs to crystallize, thereby allowing higher drug concentrations and providing increased bioavailability. However, most commercially available cellulose derivatives were not specifically designed for this application, leading to gaps in understanding the key mechanisms by which ASD operate. This creates the need for polysaccharide derivatives specifically conceptualized for ASD and for elucidating structure-property relationships. In this dissertation, I successfully demonstrated regioselective and chemoselective techniques to functionalize cellulose to prepare new ASD as well as smart tracking devices. I efficiently and successfully create complex structures via appending bile salt substituents using olefin cross-metathesis. I ascertained that high performance crystallization inhibitors can be achieved with enhanced hydrophilicity by the marriage of two classes crystallization inhibitors (cellulose and bile salts), as illustrated with the commercial, fast crystallizing prostate cancer drug, enzalutamide. I obtained ketone-functionalized cellulose derivatives using oxidation chemistry to produce fluorescent poly- and oligosaccharides (hydroxypropyl cellulose, hydroxypropyl methylcellulose, and hydroxypropyl beta cyclodextrin). Schiff-base chemistry was then explored to append a commercially available fluorescent label, Nile Blue. Due to the dynamic nature and hydrolytic lability of Schiff-bases, I applied reductive-amination chemistry with either one pot, or two-step techniques and evaluated the efficiency of these approaches. I characterized the new fluorescent polymers, and with the objective of elucidating ASD mechanisms, I investigated their response in solvents of different polarities to probe environment-sensitivity. Flavonoids are interesting drug candidates; they have been explored for many biomedical applications, including as inducers of apoptosis and functioning as antioxidants by radical scavenging. I prepared high-performance ASD polymer candidates, then prepared and characterized ASDs with different loadings of the flavonoids, genistein and quercetin. I explored the performance of polymers with different functionalities, hydrophilicity/hydrophobicity, and carboxylic acid content (cellulose acetate glutarate, 5-carboxypentyl hydroxypropyl cellulose, and hydroxypropyl methyl cellulose acetate succinate as positive control) by using in vitro dissolution studies. In this screening process, I determined that cellulose acetate glutarate provides the most advantageous enhancement, possessing the appropriate amphiphilicity to increase drug concentration in this study, supported by the similarity of the polymer and drug solubility parameters. I was further able to confirm via polarized light microscopy that advantageous nanodroplet formation occurs during the drug-release process. / Doctor of Philosophy / As sources for future ecofriendly materials, derivatives from nature offer fertile ground. One group of natural materials that attracts increasing attention to fulfill both performance and sustainability are polysaccharides, long chains of carbohydrates, that can be found in plant cell walls, exoskeletons of bugs or oceanic bottom feeders, algae, and indeed in all living things. Cellulose derivatives provide biologically safe materials that are biomedically relevant, including in the field of oral drug delivery. While most orally administered drugs are not 100% effective or absorbable, a class of drug delivery systems named amorphous solid dispersions can improve drug absorption with the aid of polysaccharide derivatives. Although amorphous solid dispersions are highly effective, there is still much room for improvement, and important opportunities to learn about the precise mechanisms that make such systems work. With fluorescent markers, I can also explore the surrounding environment of the drug delivery systems in preliminary studies. By understanding the environment of such polysaccharides, I determined important insight into how they improve oral drug availability and performance. Herein, I explored new amorphous solid dispersion polysaccharide derivatives, and how I have attached fluorescent labels to track them to learn how they work.

<p> Lambda carrageenan when injected subcutaneously causes the formation of a connective tissue granuloma. Initially there is a proliferation of connective tissue elements up to about fourteen days followed by regression so that by six weeks most collagenous tissue has disappeared and been replaced by adipose tissue. Lambda carrageenan has been identified in the granuloma by staining reactions with toluidine blue and other stains for acid polysaccharides. The present study was undertaken to localize the carrageenan by means of fluorescent antibody and autoradiography. For this purpose labelling of carrageenan by photoassimilation of 14CO2 into carrageenan was done. Different parameters affecting the incorporation of 14C into the carrageenan fractions were studied.</p> / Thesis / Master of Science (MSc)

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Glycosaminoglycans (GAGs), a large family of complex, unbranched polysaccharides, display a variety of essential physiological functions. The structural complexity of GAGs greatly impedes their availability, thus making it difficult to understand the biological roles of GAGs and structure-property relationships. A method that can access GAGs and their analogs with defined structure at relatively large scales will facilitate our understandings of GAG biological roles and biosynthesis modulation. Cellulose is an abundant and renewable natural polymer. Applications of cellulose and cellulose derivatives have drawn increasing attention in recent decades. Chemical modification is an efficient method to append new functionalities to the cellulose backbones. This dissertation describes chemical modification of cellulose and cellulose derivatives to prepare unsulfated and sulfated GAG analogs. Through these studies, we have also discovered novel chemical reactions to modify cellulose. Systematic study of these novel chemistries is also included in this dissertation. We first demonstrated our preparation of two unsulfated GAG analogs by chemical modification of a commercially available cellulose ester. Cellulose acetate was first brominated, followed by azide displacement to introduce azides as the GAG amine precursors. The resulting 6-N3 cellulose acetate was then saponified to liberate 6-OH groups, followed by subsequent (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation of the liberated primary hydroxyl groups to carboxyl groups. Finally, the azides were reduced to amines using a novel reducing reagent, dithiothreitol (DTT). Alternatively, another process utilized thioacetic acid to reduce azides to a mixture of amine and acetamido groups. Through pursuing these GAG analogs, we applied novel azide reductions by DTT and thioacetic acid that are new to polysaccharide chemistry. We systematically investigated the scope of DTT and thioacetic acid azide reduction chemistry under different conditions, substrates, and functional group tolerance. Selective chlorination is another interesting reaction we discovered in functionalization of cellulose esters. We applied this chlorination reaction to hydroxyethyl cellulose (HEC). We then utilized the chlorinated HEC as a substrate for displacement reactions with different types of model nucleophiles to demonstrate the scope of its utility. Overall, we have designed a novel synthetic route to two unsulfated GAG analogs by chemical modification of cellulose acetate. Through exploration of GAG analogs synthesis, we discovered novel methods to modify polysaccharide and polysaccharide derivatives, including azide reduction chemistry and selective chlorination reactions. Successful synthesis of various types of GAG analogs will have great potential biomedical applications and facilitate structure-activity relationship studies. / Doctor of Philosophy / Polysaccharides are long chains of natural sugars. Glycosaminoglycans (GAGs) are an important class of polysaccharides which have complicated chemical structures and play critical roles in many biological processes, including regulation of cell growth, promotion of cell adhesion, anticoagulation, and wound repair. Current methods to obtain these GAGs and GAG analogs are expensive, lengthy, and limited in capability. Novel methods to access these GAGs and their analogs would be promising and would facilitate understanding of biological activities of GAGs. Cellulose is an abundant polymer on earth and provides structural reinforcement in plant cell walls. Cellulose can be further chemically modified to tailor its physiochemical properties. Cellulose and cellulose derivatives have been widely used in many industries for various applications, such as textiles, plastic films, automotive coatings, and drug formulation. This dissertation focuses on modifying inexpensive, abundant cellulose and its derivatives to GAGs and GAG analogs. We start from the simple plant polysaccharide cellulose and obtain structurally complicated analogs of animal-sourced GAGs and GAG analogs. We reached our goal by designing a carefully crafted synthetic route, finally successfully obtaining two types of novel GAG analogs. During this process, we discovered two useful chemical reactions. We systematically investigated these chemical reactions and demonstrated their utility for polysaccharide chemical modification. These successful chemical syntheses of GAGs and their analogs will accelerate our understanding of their natural functions and have potential biomedical applications. The novel chemical methods we discovered will be helpful in chemical modification of polysaccharides.

Self-assembly of polysaccharide derivatives at liquid/solid interfaces was studied by surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Carboxymethyl cellulose (CMC) adsorption onto cellulose surfaces from aqueous solutions was enhanced by electrolytes, especially by divalent cations. A combination of SPR and QCM-D results showed that CMC formed highly hydrated layers on cellulose surfaces (90 to 95% water by mass). Voigt-based viscoelastic modeling of the QCM-D data was consistent with the existence of highly hydrated CMC layers with relatively low shear viscosities of ~ 10-3 N·s·m-2 and elastic shear moduli of ~ 105 N·m-2. Adsorption of pullulan 3-methoxycinnamates (P3MC) and pullulan 4-chlorocinnamates (P4CC) with different degrees of cinnamate substitution (DSCinn) onto cellulose, cellulose acetate propionate (CAP), poly(L-lactic acid) (PLLA), and methyl-terminated self-assembled monolayer (SAM-CH3) surfaces was also studied by SPR and QCM-D. Hydrophobic cinnamate groups promoted the adsorption of pullulan onto all surfaces and the adsorption onto hydrophobic surfaces was significantly greater than onto hydrophilic surfaces. SPR and QCM-D results showed that P3MC and P4CC also formed highly hydrated layers (70 to 90% water by mass) with low shear viscosities and elastic shear moduli. Finally, cellulose adsorption and activity on pullulan cinnamate (PC) and cellulose blend films were studied via QCM-D and in situ atomic force microscopy (AFM). The hydrophobicity of PC surfaces was controlled by adjusting the degree of cinnamate substitution per anhydroglucose unit (DSCinn). It was found that cellulase showed weak adsorption onto low DSCinn PC surfaces, whereas cellulase adsorbed strongly onto high DSCinn PC surfaces, a clear indication of the role surface hydrophobicity played on enzyme adsorption. Moreover, cellulase catalyzed hydrolysis of cellulose/PC and cellulose/polystyrene (PS) blend surfaces was studied. The QCM-D results showed that the cellulase hydrolysis rate on cellulose in cellulose/PC blend surfaces decreased with increasing DSCinn. AFM images revealed smooth surfaces for cellulose/PC (DSCinn = 0.3) blend surfaces and laterally phase separated morphologies for cellulose/PC (DSCinn ≥ 0.7) blend surfaces. The combination of QCM-D and AFM measurements indicated that cellulase catalyzed hydrolysis was strongly affected by surface morphology. The cellulase hydrolysis activity on cellulose in cellulose/PS blend surfaces was similar with cellulose/PC blend surfaces (DSCinn ≥ 0.7). These studies showed self-assembly of macromolecules could be a promising strategy to modify material surfaces and provided further fundamental understanding of adsorption phenomena and bioactivity of macromolecules at liquid/solid interfaces. / Ph. D.

The composition of the non-starch polysaccharides in sweet potato was determined and their flatulence-inducing potential evaluated. A preparative adaptation of the AOAC enzymatic method was used to isolate the insoluble and soluble non-starch polysaccharides. The insoluble non-starch fraction was mainly cellulose, but contained a substantial amount of hemicellulosic glucose, suggesting the presence of an independent glucan fraction. Relatively small amounts of xylose, galactose, arabinose, mannose, rhamnose and fucose were present. Galactose was the predominant sugar in the soluble non-starch polysaccharide fraction, although substantial quantities of arabinose and mannose were also present. Xylose, rhamnose, glucose and fucose were also present in small quantities. Uronic acids also made up a substantial portion of the soluble non-starch polysaccharides. Appreciable protein and ash were present in both the insoluble and soluble NSP fractions. The in vitro test for gas production using Clostridium perfringens as a test organism showed that both the insoluble and soluble non-starch polysaccharides are potential flatulence-inducers in sweet potato. However, the presence of the hemicellulosic glucans in the insoluble fraction make the latter a more likely candidate for flatus-inducer. / Master of Science

The relationship between substituent chemistry and melt behavior for xylan derivatives was investigated by differential scanning calorimetry and parallel-plate dynamic viscometric measurements. Xylan esters exhibit characteristic Tg values which decrease with increasing size of substituent. However, these materials do not flow at temperatures well above Tg. The ether derivative hydroxypropyl xylan was found to flow at substitution levels higher than 0.5 degree of substitution, with melt viscosity decreasing as the degree of substitution increased. The influence of viscosity ratio and composition on the texture of melt-blends of hydroxypropyl cellulose and polystyrene was studied for viscosity ratios of 0.08 to 0.55. Blends were examined by SEM and TEM. It was found that composition was the determining factor in texture, and not viscosity ratio over the range tested. Extrusion problems limited the range of compositions tested to 40% biopolymer or less. Phase inversion was not observed, although inversion was expected for some samples based on predictive models in the literature. / Master of Science