The application of carbohydrate molecules to the development of medical products and therapeutic compounds has undergone a dramatically increased level of activity in recent years, leading to the identification of “glycomics” as a legitimate new area of biotechnology.1–4 One of the most active areas of investigation are studies of glycosaminoglycans and their derivatives in developing improved anticoagulants such as new heparins.5 The N-acetyl glucosamine–containing oligo- and polysaccharides are an important class of glycosaminoglycans, some of which have been developed for medical uses such as the inhibition of surgical adhesions, the relief of joint pain caused by osteoarthritis, and the control of bleeding. Polymers containing N-acetyl glucosamine, such as chitin, chitosan, and hyaluronic acid, have been the subject of many studies.6–8 A new polymer, poly-N-acetyl glucosamine (p-GlcNAc),9,10 has been identified and found to be effective in achieving hemostasis in surgical procedures and trauma.11–13 The p-GlcNAc material is a unique polymer structure isolated and purified from large-scale cultures of a marine microalga.9,10 An investigation of the physical and chemical properties of the diatom p-GlcNAc fiber material was undertaken to determine how it differs in structure and properties from chitosan, another N-acetyl glucosamine–containing compound, to better understand prior observations of differences in hemostatic function.14
MATERIALS AND METHODS
Materials
Poly-N-acetyl glucosamine fiber suspensions and formulations were manufactured and supplied by Marine Polymer Technologies, Inc. (Danvers, MA). Chitosan samples were purchased from Sigma Chemicals, Inc.
Chemical Analysis
Analysis of test samples for chemical composition and degree of deacetylation were performed by Analytical Answers, Inc. (Woburn, MA). Methods used included scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), and carbohydrate (sugar) analysis.
Transmission Electron Microscopy
Thin membrane samples of p-GlcNAc were prepared from pure fiber slurry suspensions (approximately 1.1 mg/mL) by filtration and drying. Samples were placed on paraffin, covered with 4% glutaraldehyde fixative at 4°C, and cut into 1-mm3 slices. After storage overnight at 4°C, samples were washed four times with 0.1 mol/L Na cacodylate, 0.15 mol/L sucrose, pH 7.4 buffer. Samples were platinum coated and mounted, and microscopy was performed by standard techniques. Transmission electron microscope studies were performed by Dr. George Ruben, Dartmouth College (Hanover, NH).
Fourier Transform Infrared Spectroscopy
Thin membrane samples of p-GlcNAc were prepared from pure fiber slurry suspensions (approximately 1.1 mg/mL) by filtration and drying, and placed into the FTIR (ThermoMattson IR300) spectrometer sample chamber in the path of the infrared light beam for analysis. Spectra were collected in transmission mode at a resolution of 4 wave numbers over the mid-infrared (IR) range of 4,000 to 400 wave numbers. The absorbance band doublet at 2,340 wave numbers is attributable to atmospheric carbon dioxide.
Molecular Weight Measurement by Viscosity
Preparation of Fully Acetylated p-GlcNAc for Viscosity Measurements
Samples of p-GlcNAc were dried in an oven at 60°C. A stock solution of 1 mg/mL was made by dissolving p-GlcNAc membrane in N,N-dimethylacetamide containing 5% LiCl. The solvent was prepared as described by Terbojevich et al.15 Membranes were left stirring to dissolve overnight. After diluting the stock solution to the desired concentrations for viscometry, the solutions were stirred for 15 minutes.
Measuring the Viscosity
The kinematic viscosity of p-GlcNAc and its derivatives was measured using a Cannon-Fenske opaque viscometer. Viscometer size 75 was used to measure the viscosity of fully acetylated p-GlcNAc. Measurements were run on duplicate samples. The relative viscosity was calculated from the equation ηρ/η°ρ°, where η is the kinematic viscosity and ρ is the density. Intrinsic viscosity [η], with units cm3·g−1, was found by extrapolating the y-intercept from the graph representing the following equation:16
Circular Dichroism Spectroscopy
Thin membrane samples of p-GlcNAc were prepared from pure fiber slurry suspensions (approximately 1.1 mg/mL) by filtration and drying. Chitosan samples were prepared by drying solutions (1.0 mg/mL) to form thin membranes. Samples were placed into the circular dichroism (CD) (Cary 60) spectrometer sample chamber in the path of the ultraviolet light beam for analysis. Spectra were collected between 190 and 350 nm.
Biocompatibility Studies
Samples of p-GlcNAc fibers and membranes were provided to Toxicon, Inc. (Woburn, MA) for biocompatibility analysis. A standard series of biocompatibility assays was performed, including cytotoxicity, primary skin irritation, sensitization, systemic toxicity, hemocompatibility, pyogenicity, implantation test, mutagenicity, and subchronic toxicity.
RESULTS
Marine Polymer Technologies has developed an aseptic, bioreactor and downstream processing technology for the isolation and purification of sterile p-GlcNAc fibers from a marine microalga. Every batch of p-GlcNAc is subjected to stringent quality-control and quality-assurance assays and procedures.
Chemical Structure and Molecular Weight of p-GlcNAc
Figure 1A shows a transmission electron micrograph at 25,000× magnification of a thin membrane cast from a slurry of p-GlcNAc fibers after the manufacturing process. It can be seen that the fibers are long (often longer than 100 μm) and thin (approximately 0.5 μm). The fibers are arrayed in random orientation in the membrane shown in Figure 1A. Figure 1B shows the results of carbohydrate (sugar) analysis of the p-GlcNAc material and indicates that the fibers consist entirely of N-acetyl glucosamine sugar residues. Table 1 provides results of more than 1,000 individual chemical composition measurements. The percentage values for carbon, nitrogen, and hydrogen in Table 1 and the sugar analysis (Fig. 1B) are consistent with the chemical structure presented in Figure 2. Solid-state nuclear magnetic resonance studies were performed and are consistent with this result (data not shown).
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Fig. 1.: (A) Transmission electron micrograph of a beta–poly-N-acetyl glucosamine fiber–containing membrane at 25,000× magnification. (B) A high-performance liquid chromatography carbohydrate (sugar) analysis of the poly-N-acetyl glucosamine fiber material.
Table 1: Elemental Analysis of p-GlcNAc (Normalized Values of >1,000 Analyses)
Fig. 2.: Chemical structure of the polymer molecule found in the beta–poly-N-acetyl glucosamine fiber.
Viscosity studies were performed on samples of p-GlcNAc fibers that had been dissolved in the chaotropic solvent N,N-dimethylacetamide containing 5% LiCl. This solvent was required to solubilize the fibers because the individual polymer molecules are strongly hydrogen-bonded in the fiber structure. The viscosity data indicated that the weight-average molecular weight of the fully acetylated polymer is 2.8 × 106 Da. The p-GlcNAc fibers, therefore, consist of approximately 50 (data not shown) linear polysaccharide molecules in which each sugar residue is N-acetylated, and wherein each polymer molecule has a high molecular weight and is tightly hydrogen-bonded to others in the fiber.
Physical Structure of p-GlcNAc Fibers
Infrared (FTIR) spectroscopic studies were performed on p-GlcNAc fiber–containing thin membranes and spectra were compared with those obtained on chitosan membranes. Figure 3 illustrates that the IR spectrum of p-GlcNAc consists of very sharp, well-resolved peaks, whereas the spectrum of chitosan is diffuse and unresolved. Figure 4 includes CD spectra of p-GlcNAc and chitosan, and again the spectra are not similar. The data in Figures 3 and 4 clearly demonstrate that p-GlcNAc has a unique and different three-dimensional structure compared with amorphous randomly structured chitosan. The presence of sharp IR peaks for the p-GlcNAc fibers supports the existence of a highly ordered distinct and repetitive structure in which the individual molecules have limited motion. The broad, ill-resolved chitosan IR data suggest that there is little organized structure in those molecules.
Fig. 3.: (A) FTIR spectrum of beta–poly-N-acetyl glucosamine fiber–containing membrane material. (B) FTIR spectrum of chitosan membrane.
Fig. 4.: Circular dichroism spectra of beta–poly-N-acetyl glucosamine fiber–containing membrane and chitosan membrane.
The CD spectrum of p-GlcNAc in Figure 4 illustrates that the p-GlcNAc fibers contain polymer molecules rich in ordered acetyl groups, leading to the strong negative n-π* transition peak at approximately 218 nm. The chitosan CD spectrum, however, shows a greatly reduced magnitude and a wavelength shift of the peak to approximately 225 nm. The differences between the p-GlcNAc and chitosan CD spectra demonstrate that the two materials have a significantly different chemical structure, with chitosan having only a fraction of the acetyl groups found in p-GlcNAc, and that those groups are in a less ordered physical structural environment than the acetyl groups in the p-GlcNAc fibers.
Figure 1A is a high-resolution electron micrograph of p-GlcNAc fibers showing the long, thin nature of the p-GlcNAc fibers. Previously published data14 indicate that the N-acetyl glucosamine polymers in the fiber are oriented parallel to one another and held together in the unique beta-crystalline conformation by means of hydrogen bonding.
Biocompatibility
A set of standard biocompatibility assays were carried out (see Materials and Methods) that resulted in the determination that the p-GlcNAc is fully biocompatible. The material is being used to formulate several new hemostatic bandages, including the SyvekPatch,14 SyvekNT, and the RDH Bandage,13,17 all of which have received U.S. Food and Drug Administration clearance. These products have been tested in a large number of animal models of hemorrhage and have been shown to be efficacious in a number of clinical settings.
DISCUSSION
The microalga-derived p-GlcNAc fiber material is produced by stringent manufacturing and quality control processes. The p-GlcNAc has an excellent biocompatibility profile and can be formulated into many configurations. The chemical structure has been determined and shows that the fiber consists of fully acetylated linear polymer molecules. The p-GlcNAc fiber higher order structure has been determined by means of FTIR and CD spectral methods and by means of x-ray scattering. Its structure is an ordered and regular (beta-crystalline) configuration,14 with uniformly repeating orientation of the polysaccharide components within the fiber. This unique structure has been linked to the hemostasis performance of the product. It has been found to be a strong ligand for platelet surface receptors, including the GPIIbIIIa and GPIb proteins (T. M. Fisher et al., unpublished data). The fiber is thereby capable of activating platelets by specific receptor-based interactions, leading to a rapid expression of vasoconstrictor substances, such as thromboxane and serotonin, and also resulting in the surface exposure of phosphatidyl serine (T. M. Fisher et al., unpublished data).18 The fibers catalyze a critical step in accelerating hemostasis, the p-GlcNAc fiber mediation of fibrin polymerization. This occurs by means of an acceleration of the intrinsic coagulation pathway for thrombin generation on phosphatidyl serine-rich platelet membrane interface.
Other polymers containing N-acetyl glucosamine such as chitin, chitosan, and hyaluronic acid do not have the same structure and do not exhibit the hemostatic activity, as demonstrated by in vitro and in vivo studies (T. M. Fisher et al., unpublished data).13,14,17 There are several significant differences between p-GlcNAc and chitosan that contribute to the observed differences in the data presented above. Each individual p-GlcNAc fiber consists of approximately 50 high-molecular-weight, fully-acetylated polysaccharide molecules that are hydrogen-bonded to form the unique beta-tertiary structure determined by x-ray scattering studies. This beta-polysaccharide structure is rare in nature. The p-GlcNAc fibers are pure, with no other contaminants. The manufacture of the p-GlcNAc fibers is a proprietary, well-documented, quality-controlled cGMP process resulting in a uniform and reproducible final product. In contrast, the chitosan material is an uncharacterized mixture of deacetylated (>85%), low-molecular-weight polysaccharide components; molecular weight estimates generally indicate an average molecular weight of 40,000 Da. The manufacturing processes and quality-control and -assurance criteria for the chitosans are not known. In general, chitosans are manufactured as a waste product from crustacean shells by use of strong base and acid extraction. Treatment with strong bases results in products that have low molecular weight over a wide range of values. It is also known that the chitosans do not have a regular, stable, three-dimensional structure and thus differ significantly from the p-GlcNAc fibers in this regard. It is likely that the chitosans take on a random coil configuration when in contact with and dissolved in blood. The p-GlcNAc fibers are insoluble, given its high molecular weight and crystalline structure, and maintains its structure in the presence of blood. There is a sharp contrast between the crystalline purity and uniformity of p-GlcNAc and the chemical and molecular variability of chitosan.
CONCLUSION
It is no surprise that the p-GlcNAc fibers and chitosan differ dramatically in physical, chemical, and biological properties, given their respective natural origins. The p-GlcNAc fibers are generated by a precise molecular machinery found only in the particular microalga/diatom of its origin. The diatom is able to simultaneously synthesize a large number of high-molecular-weight (2.8 × 106 Da) polymer molecules, to assemble them into a unique fiber with a precisely ordered crystalline structure, and to extrude the fiber into the extracellular environment. This must be an active assembly process requiring considerable chemical energy, whereas the chitosan material is found in nature as a part of a complex mixture of macromolecules, including proteins, in the exoskeletons of insects and crustaceans, and in the cell walls of fungi. The two materials are quite different by all measures, and they function very differently as hemostatic agents, as has been observed.14
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