NTC Project: M05-CD01 1

Functional Fibers via Biomimesis M05-CD01 You-Lo Hsieh, Leader, ylhsieh@ucdavis.edu, Ping Lu, pplu@ucdavis.edu University of California, Davis, CA 95616-8722 Albert Abbott aalbert@clemson.edu, Michael S. Ellison ellisom@clemson.edu Clemson University, Clemson, SC 29634-1307 John Walker, Heidi Schreuder-Gibson U.S. Army Natick Soldier Center, Natick, MA 01760-5020 Goal The goal is to produce fibrous materials that incorporate enzymes and engineered proteins. Several enzymes have been chosen to represent major classes of enzymatic action, including the breakdown of oils, fatty acids, sugars, RNA and other harmful chemicals as well as those that catalyze synthesis of DNA. The focus here is to exploit chemical synthesis and fiber-engineering principles, coupled with molecular biology, to create reactive fibrous materials that are capable of binding with proteins. Several synthesis and processing methods will be explored for the development of fibers capable of containing proteins. These solid-supported enzyme proteins will be tested for preservation of their activities under common operating environments as well as after exposure to extreme conditions (temperature, pH) and extended time. Abstract This research pioneers the concept of using fibrous supports for stabilizing and improving functions, as well as identifying new and expanded applications, and recovering and conserving specialty enzymes and proteins. Several enzymes have been selected to represent structurally and functionally distinct classes of proteins to be engineered into fibrous forms. This report showed the successful entrapment of -galactosidase, the enzyme that catalyzes the breakdown of sugars, in polyacrylamide (PAAm) hydrogel nanofibers. The entrapment was confirmed by microprobe elemental mapping of sulfur present in the enzyme protein. The catalytic activity of of -galactosidase was demonstrated by X-gal essay. This project is designed to generate new biomimetic fibrous materials that contain naturally derived enzymes and engineered proteins. This research is significant in that it (1) pioneers the generation of new biomimetic functional fibers; (2) is interdisciplinary, linking biology, chemistry, and engineering; and (3) builds on knowledge generated in two highly productive NTC projects (M02-CD05 and M02-CL04). These new and specific biological functions help to meet new demands for current fibrous products as well as to build new uses and new applications of fibers. This research pioneers the concept of using fibrous supports for stabilizing and

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improving functions of specialty enzymes and proteins, as well as identifying new and expanded applications, and recovery and conservation. The results of this project will have broad applications in areas as diverse as bio-sensing and drug delivery on textile-derived substrates to high performance industrial and personal protection materials. Background One of the primary obstacles to the applications of most functional polymers and macromolecules is their lack of physical integrity or mechanical strength and flexibility for practical use. One logical solution is to reinforce these functional polymers with another material(s) via various coating, blending or entrapment mechanisms. Although studies have been extensive on various functional polymers, the generation of support matrices in particular fibrous matrices is limited. Furthermore, examples of linkages formed through chemical bonding between the functional polymers and fibers are rare. In Hsiehs laboratory at UC Davis, chemical reaction and processing techniques have been developed employing various natural and manufactured fiber/polymer matrixes as support substrates for linear and 3-D network polymers (US patent application filed in 2004). This invention successfully incorporates polymer syntheses/reactions with fiber/polymer engineering technologies to form solid-supported functional polymeric materials. We have previously produced activated natural and synthetic polymers via plasma/UV radiation and various chemical and enzymatic reaction mechanisms, such as reductive amination, propoxylation, redox, and transesterification. These activated structures were then reacted with functional compounds to generate ion-exchangers and chelators [1], amphiphilic polymers [2,3], super-absorbent [4] and stimuli-sensitive hydrogels [5], and hydrogels for controlled-release of biological agents [6-8]. Our more recent work involves the use of di- and multi-functional compounds to activate polymer surfaces. These chemical strategies provide various types of linkers or spacers with functional end groups that either have specific chemical functions or can be further reacted with other agents. In project M02-CD05, several chemical reactions and fiber formation approaches have been investigated for their proficiency to immobilize lipase hydrolases onto fibrous supports. Specifically, encapsulation and covalent bonding to solids with the aids of crosslinking and multi-functional reagents and surface-active functional reactants have been successfully developed [9,10]. Through NTC project M02-CL04, we have a demonstrated capability to produce proteins with designed functionality and to produce materials from them. This expertise is apropos the current project in that we are able to design functional proteins for fibrous support coating. This current project furthers the successes attained by M02-CL04 in the formation of materials through the electrospinning of designer proteins. Progress Several enzymes have been selected to represent structurally and functionally distinct classes of proteins to be engineered into fibrous forms. They include those that catalyze the breakdown of

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sugars (-galactosidase), the synthesis of DNA (Teg polymerase) and the breakdown of RNA (Ribonuclease A or RNAnase). Other biomolecules we will consider include enzymes that break down oils and other chemicals. The major focus is to understand protein-polymer and proteinfiber interactions for exploring feasible mechanisms of incorporating proteins as part of the fiber structures, while maintaining the functions of the proteins. Most enzymes are unstable under ambient conditions and degrade under elevated temperature and exposure to organic environments. Protecting these enzyme proteins via chemical modification is to be evaluated to see if this would stabilize proteins against degradation by organic solvents or high temperatures. To date, we have made significant progress toward 1) generating fibers to encapsulate galactosidase; 2) establishing assays for the qualitative and quantitative determination of galactosidase; and 3) strategizing other approaches for both -galactosidase and Taq polymerase. -galactosidase catalyzes the breakdown of lactose and other -galactosides into monosaccharides. -galactosidase is a tetramer consisting of four equal subunits of 135,000 each. It is a sulphydryl containing enzyme, with 19 cysteine residues per subunit. Some of these are present in disulfide bridges. The number of active sites per molecule of enzyme is thought to be one per monomer of 135,000. Histidine residues are believed to be present in the active site of -galactosidase. The SH groups are thought to be important in maintaining the active conformation of the enzyme, therefore are to be avoided in the consideration for binding to fibers.

Of the immobilization mechanisms of entrapment, adsorption and covalent bonding, entrapment is the best strategy for -galactosidase due to its shear size (Table I). This is particularly true when the solids to embody these proteins are sub-micrometer size fibers. The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. It is done in such a way as to retain the protein molecules while allowing penetration of the substrate.

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Table I. Enzymes dimensions

Entrapment of -galactosidase has been incorporated into the non-ionic poly(acrylamide) (PAAm) matrix in the fiber formation electrospinning process. PAAM is readily soluble in water. The nonionic PAAm do not form fibers easily at either high (5,000 kDa) or low (10 kDa) molecular weights. The specific viscosity of aqueous solutions of 5,000 kDa PAAm increased drastically with concentrations, from 5.7 to 17563 with concentrations from 0.5wt% to 3.5wt%. The logarithm-logarithm plots of specific viscosities (sp) of PAAm solutions as a function of PAAm concentrations show two changes in the slope (Figure 1), showing the onset of semidilute entangled regime (Ce=1.0wt%) and concentrated regime (C**= 2.7wt%). The concentrations below 1.0wt% are semidilute unentangled (sp ~ 2.51), and concentrations between 1.0wt% and 2.7wt% are semidilute entangled (sp ~ 3.64) , and concentrations above 2.7wt% are concentrated (sp ~ 9.29).
10000

Specific Viscosity

1000

PAAm5000

PAAm5000/PAAm10

100

PAAm10
10

1wt%

10 18.7wt% 2.7wt% 32.9wt% 10.6wt% Concentration (wt%)

Figure 1. Viscosities of aqueous solutions of PAAm at 5,000kDa (PAAm 5000), 10kDa (PAAm10), and their mixtures (PAAm 5000/PAAm10) at varying concentrations. Uniform fibers could be generated from the 5,000 KDa PAAm at 2.5% concentration (Figure 2a), while substantial beaded structures were observed at lower concentrations. While the low molecular weight PMMa (10 kDa) was not fiber-forming (Figure 2b), its addition to the 2.5% aqueous solution of the 5,000 kDa PAAm improved the efficiency of fiber production. Mixing of the two chain lengths of PAAm appeared to allow sufficient entanglement yet efficient flow to be fiber-forming. Fibers increase in size with increasing PAAm quantities (Figure 3)

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a b Figure 2. SEM of fibers electrospun from aqueous solutions of: (a) 2.5wt%.of PAAm 5,000 kDa (b) 50wt% of PAAm 10 kDa

a b c Figure 3. SEM of PAAm fibers electrospun from aqueous solutions with constant 2.5wt% PAAm5000 and varied PAAm10: (a) 0wt% (b) 5wt% (c) 10wt%. These unique nanofibrous PAAm membranes were very unstable in an aqueous environment, i.e., turning into clear, gelatinous substrate immediately and then dissolved rapidly. Therefore, chemical cross-links among the linear PAAm chains in the fibers were deemed necessary. The PAAm nanofibrous membranes were cross-linked with 10 wt% glutaraldehyde (GA) in the presence of 4 wt% of HCl. Under optimized conditions, the cross-linked PAAm nanofibrous membrane exhibited excellent shape and structure retention following prolonged water immersion (72h) (Figure 1a). Not only do these fibrous membranes remain hydrophilic and retain superior water absorbency (Figure 1b), the swelled fibers returned to their original forms. These crosslinked PAAm fibrous membranes could be easily handled both in water and out of water. Furthermore, the cross-linked PAAm nanofibrous membranes were also insoluble in common organic solvents. In fact, these nanofibrous membranes show no structural change after 72h exposure to methanol, ethanol, acetone, chloroform, DMF, and cyclohexane, excellent organic stability.

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Figure 4. Cross-linking of PAAm by 10 wt% glutaraldehyde in EtOH with 4 wt% HCl at 120oC for 60 min and followed by immersing in water for 72 h (a) SEM and (b) image in water. -Galactosidase is readily available from commercial sources, e.g, Sigma Biochemicals and Reagents #48275 or G-5635. The PAAm fibrous membranes were immersed pH 7 buffer solution containing -Galactosidase over 24h. The SEM image of the dried PAAm membrane showed complete structure retention. The fibers remained smooth with no observable enzyme aggregates on fiber surfaces or inter-fiber pores (Figure 5). This suggests internal entrapment of -Galactosidase.

Figure 5. SEM micrographs of -galactosidase entrapped PAAm nanofibrous membranes (PAAm: ~50 mg; -galactosidase: ~2 mg/ml; 1 ml; pH 7; 4oC for 24 h). The entrapment and presence of -galactosidase in the PAAm fibrous membranes was confirmed by microprobe elemental mapping of sulfur present in the 76 cysteine units in the enzyme structure. The sulfur mapping of the cross-linked PAAm blank only exhibited a small amount of weak noises which were mainly caused by the error of the instrument (Figure 6a). The sulfur signals of the -galactosidase entrapped PAAm nanofibrous membranes were intense and evenly distributed (Figure 6b). The -galactosidase molecules were thought to be entrapped inside the PAAm network. These intermolecular pores inside the cross-linked PAAm network expand

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when swollen in aqueous buffer and open to allow diffusion of the enzyme molecules. Upon reaching equilibrium and drying, the enzyme molecules are entrapped internally. a b

Figure 6. Sulfur mapping of cross-linked PAAm (a) blank and (b) sample by electron microprobe. The activity of -galactosidase can be monitored using a variety of chromogenic and fluorogenic substrates. A qualitative method using 5-Bromo-4-chloro-3-indolyl -D-galactoside (X-gal) has been employed to detect the presence of -galactosidase. X-gal is a galactose sugar with a glycosidic linkage to a chromophor and stays colorless, until the glycosidic link is broken by hydrolytic action of -galactosidase to release the blue chromophor. The essay to quantify galactosidase involves spectrophotometric measurement of the formation of o-nitrophenol (ONP) as the hydrolytic product of the action of -galactosidase on o-nitrophenyl -D-galactoside (ONPG). X-gal essay was conducted to further confirm the entrapped enzyme and its activity. After 24 h incubation, the sample containing the entrapped -galactosidase turned blue while the blank still remained clear (Figure 7). The color change not only confirmed the existence of -galactosidase but also demonstrated that catalytic activity was retained.

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Blank

Sample

Fig. 5. Assay of -galactosidase by 20 mg/ml x-gal in DMF (~10 mg membrane immersed in 1 ml pH 7 buffer solution and incubated at 37oC for 24 h). Future work includes the use of strong secondary forces, such as ionic interaction, and covalent bonds to tether enzyme molecules on ultra-fine fiber surfaces. Another major enzyme, RNase A, will be targeted for immobilization via both adsorption and secondary force mechanisms. Taq polymerase catalyzes the synthesis of DNA. Polymerases are enzymes that replicate or make new DNA. The technique to produce new DNA is called polymerase chain reaction or PCR. It was developed by Nobel laureate biochemist Kary Mullis in 1984 based on the discovery of the biological activity of DNA polymerases found in thermophiles (bacteria that live in hot springs). Taq polymerase, named after the bacteria Thermus aquaticus, is derived from a thermophilic DNA polymerase. Most DNA polymerases work at low temperatures at which DNA molecules are tightly coiled and cannot be accessed by the polymerases. But at 100oC, the temperature the thermophile DNA polymerases function, DNA is denatured (in linear form) and becomes more accessible. Half-life of Taq polymerase is 1.6 hours at 95oC. At 37oC, Taq polymerase has only about 10% of its maximal activity. PCR allows the replication, thus the amplification of a small amount of DNA into a larger amount for more accurate and reliable detection and measurements. Ribonuclease A (RNase A) catalyzes the hydrolysis of single-stranded RNA by cleaving the phosphodiester bond. RNase A is a single chain polypeptide containing disulfide bridges. RNase A exhibits activity from 15-70oC with the optimal temperature for activity at 60oC. The pH optimum is 7.6, with an activity range of 6-10. RNase A is also a very stable enzyme and can withstand temperatures up to 100oC. At 100oC, RNase A is most stable between pH 2.0 and 4.5. RNase A can be inhibited by alkylation of the histidine-12 or histidine-119 which is present in the active site of the enzyme. These should be included in the consideration for binding as well. Activators of RNase A include potassium and sodium salts. The highest activity is exhibited with single stranded RNA. RNAase A is available from Sigma Biochemicals and Reagents (#R-4875 for cruder, # R-5125 for pure).

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Regarding generating new biomimetic fibrous materials that contain engineered proteins, tissue engineering scaffolds with synthetic spider silk proteins were specifically targeted owing to its protein reactivity. Synthetic spider silk protein, i.e., Spidroin 1-collagen copolymer, was provided by Dr. Albert Abbott of the Genetics and Biochemistry Department, Clemson University. The Spidroin 1-collagen copolymer protein was expressed by engineered yeasts with spidroin 1-collagen genes. Details are available in the Ph.D. dissertation of Florence Teul [Clemson University, 2004]. For tissue engineering, the method by which the biomaterials are fabricated into scaffolds is important for achieving an appropriate morphology. Electrospinning has the potential to process polymers into porous scaffolds comprising submicron fibers. The fiber-formation process of electrospinning requires a polymer solution with high enough viscosity and surface tension to prevent the jet from breaking up before reaching the target. The viscosity and surface tension of spider silk protein solution by itself was too low to satisfy this requirement; thus, a viscosity modifier, poly(vinyl alcohol) in this case, was added to the solution to induce a suitable viscosity and surface tension. The electrospinning of synthetic spider silk protein/poly(vinyl alcohol) solutions successfully generated fibrous structure as shown by the SEM (Figure 4, next page). The chemical structure of the synthetic spider silk protein appears to be preserved as evidence by the FTIR results. Electrospun SSP/PVA mats were compared with electrospun PVA mats for their ability to act as tissue engineering scaffolds for fibroblasts. Results indicated that the addition of silk protein to PVA did not enhance cell attachment and growth on the electrospun scaffolds, perhaps because of the low concentration of the spider silk proteins in the scaffolds. The interaction between fibroblasts and scaffolds was mainly because of the PVA. More concentrated synthetic spider silk protein solution is needed to shed light on this issue. Details of this work can be found in the M.S. thesis of Jing Xu [Clemson University, 2006].

Figure 4. Effect of Methanol Treatment on the Morphology of Mats Electrospun from Solutions of PVA and PVA with Spidroin Protein. First row: PVA (10%); second row PVA (10%) and protein (2%). First Column: before MeOH; second column, after MeOH.

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Outreach These investigators seek collaboration with other researchers from academia as well as industry to develop fibrous supports containing various proteins and to identify enzymes of specific industrial interest. Collaboration with the government is anticipated. In the long run, industrial partners from the polymer/fiber/textile industries as well as the biotechnology and biomedical sectors will be sought for further development of specific protein-fiber applications and for the transferal of the developed technology to all affiliated industries, linking university and industrial partners. Project Website: http://www.ntcresearch.org/projectapp/?project=M05-CD01 Students: Ping Lu (Ph.D., UC Davis) and Jing Xu (M.S., Clemson University) References: 1. Lin, W.P. and Y.-L. Hsieh. Kinetics of Metal Ion Absorption on Ion-exchange and Chelating Fibers, Industrial and Chemical Research 35(10): 3817-3821 (1996). 2. Zhou, W.J., M.E. Wilson, M.J. Kurth, Y.-L. Hsieh, J.M. Krochta, and C.F. Shoemaker. Synthesis and properties of a novel water-soluble lactose-containing polymer and its crosslinked hydrogels, Macromolecule, 30(23): 7063-7068 (1998). 3. Lin, W., M. Hu, Y.-L. Hsieh, M.J. Kurth, and J.M. Krochta, Thermo-sensitive lactitol-based polyether polyol (LPEP) hydrogels, Journal of Polymer Science, Polymer Chemistry Edition, 36:979-984 (1998). 4. Zhou, W.-J. , M.J. Kurth, Y.-L. Hsieh, J.M. Krochta, Synthesis and thermal propeties of a novel lactose-containing poly(n-isopropylacrylamide-co-abrylamidolactamine) hydrogel, Journal of Polymer Science, Polymer Chemistry 37:1393-1402 (1999). 5. Zhou, W.-J. , M.J. Kurth, Y.-L. Hsieh, J.M. Krochta, Synthesis and characterization of new styrene main-chain polymer with pendant lactose moiety through urea linkage, Macromolecule 32(17): 5507-5513 (1999). 6. Chacon, D., Y.-L. Hsieh, M.J. Kurth, and J.M. Krochta, Swelling and Protein Absorption/Desorption of Thermo-Sensitive Lactitol-Based Polyether Polyol (LPEP) Hydrogels, Polymer 41: 8257-8262 (2000). 7. Han, J. H., J. M. Krochta, M. J. Kurth and Y.-L. Hsieh. Lactitol-based poly(ether polyol) hydrogels for controlled release chemical delivery systems. J. Agric. Food Chem 48(11): 5278-5282 (2000). 8. Han, J. H.., J. M. Krochta, M. J. Kurth and Y.-L. Hsieh. Mechanism and characteristics of protein release from lactitol-based crosslinked hydrogel. J. Agric. Food Chem. 48(11): 5658-5665 (2000). 9. Wang, Y. and Y.-L. Hsieh, Enzyme immobilization to ultra-fine cellulose fibers via amphiphilic polyethylene glycol (PEG) spacers, Journal of Polymer Science, Polymer Chemistry , 42:16, 4289-4299 (2004). 10. Chen, H. and Y.-L. Hsieh, Enzyme immobilization on ultra-fine cellulose fibers via poly(acrylic acid) electrolyte grafts, Biotechnology and Bioengineering 90(4): 405-413 (2005).

National Textile Center Annual Report: November 2007