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`Page 2
`
`
`
`
`
`
`

`

`34
`
`Practical Aspects of Hyaluronan Based Medical Products
`
`HCl as media for DMAB
`
`—
`
`Hyaluronic acid with
`N-acetylgluwsamine
`at the reducing and
`
`OH' b0"
`
`‘ N'
`
`DMAB ’
`
`HCI
`
`do
`Chromogensl and II
`monoanhydro
`sugar
`
`*0
`Chromogen |l|
`furan derivative
`
`\ +/NI
`
`‘—’
`
`0
`
`Ho\
`
`/N|H
`
`0
`
`Red colored substance with maximum absorption at 586 nm
`as proposed by Muckenschnabel et al.
`
`FIG U RE 2 .8
`
`The furan-type chromogen intermediate formed by the reducing-end N—acylhexosamine and
`the formation of red color during Morgan Elson reaction — findings by Muckenschnabel et al.
`
`of chromogen III and DMAB. On the basis of the data from HPLC, UV-Vis,
`and LC-MS, the authors proposed the structure of the red substance as
`mesomeric forms of N-protonated 3-acetylimino-2-(4-dimethylaminophenyl)
`methylidene-S-(l,2-dihydroxyethyl)furan (Fig. 2.8).
`Although the detailed mechanisms of the chromogenic reactions (Dische,
`Elson-Morgan, Morgan-Elson, and Reissig) may not be exactly clear for
`general consensus, the common characteristics of those reactions appear to
`involve a furan derivative and an aldehyde.
`
`——
`
`2.3 Chemistry of HA Degradation
`
`2.3.1 Alkaline Degradation
`
`The a—hydrogen on the carbon atoms adjacent to carbonyl group is labile
`under alkaline conditions. This chemical property is by and large responsible
`for the degradation of HA when trFeategl with strong alkali such as NaOH
`age
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 3
`
`

`

`Chemistry
`
`35
`
`COOH
`
`no
`
`H
`
`1|
`
`0 H0
`
`OH
`
`CH20H F'OH
`OHH
`
`0 —’ R HO
`NHAc
`
`1
`
`COOH
`
`0H
`
`0H
`
`0
`
`\NaBH4
`
`COOH
`
`F!
`
`HO
`
`'OH/z/
`
`CH20H
`OH H
`
`OH
`
`NHAc
`
`O H
`
`OH
`
`FIG U RE 2 .9
`
`Progressive degradation of hyaluronic acid from the reducing end — ”peeling reaction” — may
`be prevented by treatment with NaBH‘
`
`The N-acetylglucosamine of HA at the reducing end of the polymer chain
`exists as hemiacetal and aldehyde in equilibrium. The a-hydrogen of the
`aldehyde is reactive, and the neighboring glycosidic bond is prone to
`cleavage under strong basic conditions. The reaction would peel off the
`N -acetylglucosamine unit and expose the resultant glucuronate at the
`reducing end to further degradation. Such unintended progressive erosion
`of the polysaccharides from the reducing end can be prevented by the addi-
`tion of NaBH4. The reducing agent NaBH4 reduces the aldehyde group to
`an alcohol group, rendering the neighboring hydrogen atom inactive, and
`thus preventing the stepwise degradation of the polysaccharide from the
`reducing end (Fig. 2.9) .2132 However, for HA, the a—hydrogen of the aldehyde
`group at the reducing end is not the only labile hydrogen atom in the giant
`molecule. For every repeating disaccharide unit of HA, there is a labile H-atom
`at the 5 position of the glucuronic acid ring.
`During the investigation of the colorimetric reactions of N-acetylglu-
`cosamines, Aminoff found that when HA was treated with hot dilute alkali
`or alkaline buffers, a substance was formed with maximum ultraviolet (UV)
`absorbance at 230 nm. This was observed by Kuo23 in his study to deacetylate
`HA under basic conditions, similar to the deacetylation of colominic acid
`reported previously in the literature”!25 Thus, HA with a molecular weight
`(MW) of approximately two million was dissolved in sodium hydroxide
`containing 1 mg /ml NaBH4, sealed in glass tubing, and heated at 105—110°C
`for 2 hours. Then the solution was dialyzed against flowing water for 2 days
`in a dialysis bag with a 12,000 MW cutoff. After lyophilization, the recovery
`of the treated HA turned out to be less than 2%. It is apparent that significant
`Page 4
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 4
`
`

`

`36
`
`Practical Aspects of Hyaluronan Based Medical Products
`
`HO 0
`
`0
`
`.
`
`
`NaOH, 105°C
`
`no
`
`HO
`
`FIGURE 2.10
`
`OH
`
`‘¥OH OH
`
`N80
`
`HO
`
`0
`/ O
`
`OH.
`
`OH
`
`Hyaluronic acid undergoes B—elimination when treated with hot alkali and forms an a, B
`unsaturated carboxylate disaccharide that has a strong ultraviolet absorbance at 232 nm.
`
`degradation occurred during the reaction, and the degradation probably did
`not just occur from the reducing end.
`The UV spectrum of the degraded HA product showed a strong absor-
`bance at 232 nm, which is characteristic of 0c, [3 unsaturated acid, as suggested
`by Linker et al.26 Under the reaction conditions described, HA may have
`degraded at the 1—)4 glycosidic linkages through B-elimination (Fig. 2.10).
`
`2.3.2 Acidic Degradation
`
`Glycosaminoglycans (GAGs) in general have considerable resistance to acid
`hydrolysis and its degradation under such conditions is rarely complete.
`Certain structural features of GAGs have a significant effect on the rate of
`acid hydrolysis. When the N—acetyl group is intact, the glycosidic bonds are
`readily hydrolyzed. As the deacetylation occurs simultaneously with the deg-
`radation of glycosides, the freed amino groups make the glycosaminide
`bonds more resistant to acid hydrolysis.27 Moggridge and Neuberger studied
`the kinetics of acid hydrolysis of methylglucosaminide and explained that
`the inhibition of its hydrolysis may be a result of ”the effect of adjacently
`positively charged amino-group in repelling hydrions from its immediate
`vicinity. ”23
`In the study of the chemistry of HA degradation, the focus of interest has
`been on the nondestructive depolymerization reactions that produce discrete
`HA oligosaccharides. Inoue and Nagasawa prepared even-numbered HA
`oligosaccharides from high MW (2M Da) by acid hydrolysis?-9
`Marchessault and Ranby proposed an induction-stabilization theory of the
`effect of uronic acid on the neighboring glycosidic bonds.30 BeMiller
`described such effect using carboxylcellulose as an example.31 The presence
`of an electronegative carboxyl group at the C-5 position can exert inductive
`influence on the glycosidic oxygen atoms in two different ways. It facilitates
`the electron shift toward the glycosidic oxygen (making it more labile) that
`is closer to the carboxyl group, while opposing the electron shift to the
`glycosidic oxygen (making it more stable) that is situated farther toward the
`other side of the pyranoside ring (Fig. 2.11). If the same mechanism applies
`to HA, it could well explain why the even-numbered HA oligosaccharides
`with the glucuronic acid at the nonreducing ends become the predominant
`products when HA is treated with thdrsochloric acid.29
`age
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 5
`
`

`

`Chemistry
`
`37
`
`CHZOH
`
`o w o
`
`CH2OH
`
`0
`
`A
`
`OOOH
`
`a
`
`0
`
`OH
`
`OH
`
`OH
`
`FIG URE 2.1 1
`
`The carboxyl substituent at C6 of alternate disaccharide rings of cellulose may have an inductive
`effect on the electron shifts of neighboring glycosidic bonds. It favors electron shift from ring
`A to B but opposes the shift from ring B to A.
`
`From a stereochemistry standpoint, the carboxyl group may serve as pro-
`ton donor to the closest glycosidic oxygen — normally a first step toward
`depolymerization — and also serve as an intramolecular nucleophile to
`facilitate the breaking of the glycosidic bond, as also suggested by Roy and
`Timell and by Bochkov and Zaikov (Fig. 2.12)?”3
`
`2.3.3 Oxidative Reductive Depolymerization
`
`HA can undergo oxidative reductive depolymerization (0RD), in which both
`oxygen and reducing agents are involved. According to Pigman and Rizvi,34
`the reducing substances often used are ferrous or cuprous ions and ascorbic
`acid, as well as sulfhydryl (-SH)-containing organic compounds like cys-
`teine and glutathione. Ferrous ions are the most effective reducing sub-
`stances. Phosphate ions greatly speed up the reaction compared to the
`chloride ions. At 30°C and pH 7.3, the specific viscosity of HA could drop
`90% in a time as short as 5 minutes. Synovial fluid showed similar degra-
`dations. The reaction is inhibited by glucose and completely repressed by a
`high concentration of alcohol.
`Although the ORD reaction can degrade polysaccharides and nucleic acid
`in general,35 evidence indicates that a glycosidic linkage near a hydroxyl
`group of a uronic acid polymer may be particularly susceptible.34
`
`
`
`Cellobiouronic acid
`
`FIG URE 2.1 2
`
`A carboxylic group close to a glycosidic bond can be an effective intramolecular proton donor
`at pH 2 pKa. The nucleophilic attack by the carboxylate may cause the decomposition of the
`oxoniurn ion, thus splitting the glycosidic bond.
`Page 6
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 6
`
`

`

`38
`
`Practical Aspects of Hyaluronan Based Medical Products
`
`The degree of degradation of HA by different ORD systems varied signif-
`icantly. Swann treated HA with ascorbic acid alone, which reduced the MW
`of HA from over a million to 65,000 Da.36 Cleland et al. reported the degra-
`dation of HA to its fragments with average MW of 24,000 under similar
`conditions.37 Uchiyama et al. conducted an 0RD reaction of HA by using
`Fe(ll) in phosphate buffer and under an oxygen atmosphere. The starting
`HA had an average MW of approximately 400,000, and 0RD reaction con-
`verted it to HA fragments within the range of 1000 to 10,000 .33 This confirms
`the conclusion by Pigman that Fe(]1) is among the most effective reducing
`substances for ORD reactions,34
`
`The oxidative effect of ascorbic acid in the degradation of HA is largely, if
`not exclusively, caused by the metal catalysis, which can exist as a contaminant
`in phosphate buffer and HA itself. At millimolar concentration, ferrous or
`cuprous ions cause depolymerization. At micromolar concentration, they are
`inactive alone but accelerate the rate of depolymerization by ascorbic acid.
`Ascorbic acid acts to regenerate ferrous ions by the following mechanismz39
`
`Ascorbic acid + Fe(I]I) —> dehydroascorbic acid + Fe(]1)
`
`HA + H++ Fe(II) + 02 —> degraded HA + Fe(III) + H20
`
`Superoxide free radical 02" is formed during the autoxidation reaction
`Fe(ll) —) Fe(Ill).4°'41 The free radical 02" can also be formed by the action of
`certain oxidative enzymes such as xanthine oxidase. Accompanying the
`production of the superoxide is the production of hydrogen peroxide, as the
`latter is the reaction product of the former through dismutation.42
`
`02" + 02" + 2H+ —) 02+ H202
`
`The superoxide radicals and the hydrogen peroxide further react to produce
`the hydroxyl radicals OH'
`
`02" + H202 —> 02 + OH— + OH'
`
`McCord found that both 02" and H202 were necessary for degradation
`of HA to occur. Superoxide dismutase is a scavenger for 02". Catalase
`promotes the conversion of hydrogen peroxide to water and molecular
`oxygen. The addition of either superoxide dismutase or catalase protected
`HA against the free-radical degradation. McCord42 concluded that the
`hydroxyl radicals OH' are ultimately responsible for the degradation of HA
`in the ORD reaction.
`
`Hydroxyl radicals can be created at the sites of inflammation where
`polymorphonuclear leukocytes may be activated by immune complexes
`and other opsonized material to produce oxygen-derived free radicals.
`These free radicals are capable of oxidizing tissue components to cause
`irreversible damage. This process can be inhibited by sufficient amount of
`superoxide dismutase.42
`
`Page 7
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 7
`
`

`

`Chemistry
`
`TABLE 2.1
`
`39
`
`Structures of Degradation Products from the Oxidative Reductive
`
`Depolymerization Reaction of Hyaluronic Acid
`
`Fragments from Reducing End
`
`Fragments from Nonreducing End
`
`4,5-unsaturated GlcUA(Bl—>3)N-aoetyl-D—
`glucosaminic acid (21%) (see Structure B)
`
`4,5-unsaturated GlcUA(Bl—)3)GlcNAc (Bl—)3)—D-
`arabo-pentauronic acid (24%) (see Structure A)
`
`L—threo—tetro—dialdosyl-(1—>3)
`GlcNAc (a tentative structure, 8%)
`(see Structure C)
`
`N-acetylhyalobiuronic acid (20%)
`
`N—acetyl-D—glucosamine (51%)
`
`N-acetyl-D-glucosamine (45%)
`
`Uchiyama et a1. investigated the mechanism of 0RD reactions and the
`structures of its degradation products in great detail.38 HA was first degraded
`to its fragments, with an average MW of 2600 Da by the actions of Fe(II) and
`oxygen. Those fragments were then digested with chondroitinase AC-II. The
`final reaction products as oligosaccharides and monosaccharides were sep-
`arated by gel filtration and ion-exchange chromatography, and their struc-
`tures were determined by NMR (proton and carbon-13) and fast—atom
`bombardment mass spectrometry.38 The results of the analysis of the final
`degradation products are listed in Table 2.1, and some of their structures are
`illustrated in Fig. 2.13. The study concluded, ”the 0RD reaction of hyalur-
`onate proceeds essentially by random destruction of unit monosaccharides
`
`OOOH
`o
`
`/OH
`
`OH
`
`0
`HO
`
`CHZOH
`/—0
`
`OH
`NHAc
`
`OOH
`
`HO
`
`OH
`
`(a)
`
`COOH
`/ 0
`OH
`
`0H
`
`HO
`
`(b)
`
`CHZOH
`OH
`
`OOOH
`
`NHAc
`
`CHZOH
`
`0H
`
`HO
`0
`OHC\(‘)H_/*OH
`
`0H
`
`NHAc
`
`(°’
`
`FIGURE 2.13
`
`Structures of some degradation products from the oxidative reductive depolymerization reac-
`tion of hyaluronic acid: (a) 4,5-unsaturated GlcUA(Bl—3)GlcNAc(Bl—3)—D—arabo-pentauronic ac—
`id, (b) 4,5-unsaturated GlcUA(B1~3)—N~acetyl~D—glucosaminic acid, and (c) L—threo—tetro-
`dialdosyl- (Bl-3)GlcNAc.
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 8
`
`Page 8
`
`

`

`40
`
`Practical Aspects of Hyaluronan Based Medical Products
`
`due to oxygen-derived free radicals, followed by secondary hydrolytic cleav-
`age of the resulting unstable glycosidic substituents."
`
`2.3.4 Degradation on Freeze Drying
`
`Degradation of HA can occur during freeze drying. Wedlock et al. reported
`that free-radical scavengers such as Cl‘, 1‘, alcohols, and sugars could
`inhibit this degradation.43 It was concluded that freeze drying of NaHA
`caused mechanical stress and induced free carbohydrate radicals that depo-
`lymerized the polysaccharide. The produced hydroxyl radicals during
`freeze drying is attributable to carbohydrate radicals derived from C-C
`bond cleavage.43
`Tokita et a1. studied the degradation of HA in freeze-drying conditions by
`experimental methods such as electron spin resonance (ESR) and theoretical
`calculation of molecular dynamics. The researchers observed that freeze
`drying generates three times the amount of carbohydrate radicals from the
`acid form of HA as compared to the free radicals generated from the salt
`form of HA. Meanwhile, molecular dynamics calculation revealed three
`types of water molecules captured in the vicinity of HA molecules: (1) near
`the 1-4 glycoside pocket are the first type of water molecules that associate
`with the glycoside oxygen and an N-acetyl group through hydrogen bond-
`ing, as well as interacting with a carboxyl group through Coulombic force;
`(2) in the pocket near the 1-3 glycoside linkage are the second type of
`water molecules bonded to a glycoside bond, two hydroxyl groups, and
`an N—acetyl group; and (3) in the vicinity of carboxyl groups, water molecules
`are weakly captured through Coulombic force and observed only in the case
`of NaHA — not its acid form. Water molecules of types 1 and 2 are more
`tightly bond to the HA molecules and are named nonfreezing molecules,
`whereas water molecules of type 3 are more loosely associated with HA mole-
`cules, and so are named freezing water or free water. The authors concluded
`that water contributes to the stability of HA; the HA—H chain without water
`tends to be more mobile, and therefore tends to generate more free radicals,
`than the HA-Na chain without water,- and HA-H also has higher reactivity
`toward the hydroxyl radicals as well.44
`
`2.3.5 Radiolysis
`
`It has long been known that HA solutions decrease viscosity when exposed
`to UV radiationfif’l“ A significant drop of viscosity of HA occurred even
`when the radiation dose was as low as 50 rads.47
`
`Caputo reported in 1957 the depolymerization of HA by x-rays.“"3 The
`irradiation of HA extracted from umbilical cord was carried out with soft
`
`x-rays (the wavelength range for x-rays is from about 1&8 m to about 1011 m,
`and the corresponding frequency range is from about 3 x 1016 Hz to about
`3 x 1019 Hz; hard x-rays are of higher frequency and are thus more energetic,
`whereas the lower-frequency x-rays are called soft x-rays). Sedimentation
`Page 9
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 9
`
`

`

`Chemistry
`
`41
`
`constant and electrophoretic patterns of the radiated HA were used to assess
`the degree of its depolymerization. The effect of irradiation (200 x 103 rads)
`on the degradation of HA was almost as dramatic as that of a hyaluronidase
`treatment. It was also found that the quantity of glucosamine as measured
`by Elson Morgan assay was directly proportional to the dose of x-ray. There-
`fore, it was concluded that the ”ionizing radiations, like hyaluronidase, act
`on HA by opening the N—acetylglucosaminic bond?“
`Balazs et al. studied the degradation of HA exposed to gamma irradiation
`(gamma radiation is high-energy photon emission resulting from natural
`radioactivity and is the most energetic form of electromagnetic radiation,
`with a very short wavelength [less than 1010 m] and high frequency [greater
`than 1018 Hz]). Electron paramagnetic resonance was used to analyze the deg-
`radation products. The study concluded that ”both glycosidic cleavage and
`attack of the C—5 hydrogen in the pyranose ring may take place to give the
`transient free radicals.”7 The exposure of HA to electron beam radiation
`with doses ranging from 1 to 4 x 106 rads not only reduced the molecular
`size of HA but also destroyed the chemical structures of hexosamine and
`hexuronic acid.46
`
`The fact that HA is so susceptible to very low dose exposure to radiation
`has practically ruled out the use of radiation as a sterilization method for most
`HA—based viscoelastic products. According to Remington’s Pharmaceutical Sci-
`ences, ”radiation doses of 15 to 25 kGy are sufficient to kill the most resistant
`microorganism/1"9 This is tantamount to the dose at mega rad (1 Mrad =
`10 kGy) level, which is thousands times the dose that a typical HA polymer
`formulation can withstand.
`
`2.3.6 Heat Degradation
`
`It is well known that HA, especially when in the form of an aqueous solution,
`cannot withstand elevated temperatures for any significant amount of time.
`Many have observed the dramatic decrease of viscosity of HA solutions
`when subjected to conditions of autoclaving (e.g., 121°C for 12 minutes). The
`degradation product shows a strong UV absorbance at 232 nm, strongly
`indicating the breaking of glycosidic bonds through elimination reaction and
`the formation of a, Bunsaturated carboxylate.
`Lowry and Beavers investigated the thermal stability of NaHA in aqueous
`solution within a temperature range from 25° to 100°C.49 NaHA with an
`average MW of 1.3 million Da was formulated into a 0.03% (wt/wt) solution at
`neutral pH. Samples sealed in ampoules were exposed to the set tempera-
`tures for up to 96 hours. Cannon-Fenske Routine Viscometers were used to
`measure capillary flow times (cft). The ratio (r) of the capillary flow times
`of the sample with certain exposure time (cft.) over initial sample cfto, that
`is r = cftt/cfto, is an indication of the degree of decrease of the viscosity of
`NaHA samples under the given condition.
`The study indicated that at 25°C, the NaHA solution was stable, and a
`drop of viscosity of 10% would require many thousands of hours according
`Page 10
`
`© 2006 by Taylor & Francis Group, LLC
`
`Page 10
`
`

`

`42
`
`Practical Aspects of Hyaluronan Based Medical Products
`
`to the extrapolation of the acquired data. As a contrast, a 10% drop of
`viscosity occurred in less than an hour at 90°C. The decline of viscosity over
`the time started to accelerate exponentially at 60°C. A particularly interesting
`observation of the study was that all decreases in the viscosity of NaHA
`samples started with an initial but transient increase of viscosity, as the
`samples were exposed to the heat. It was suggested that the higher temper-
`ature induces separation of HA chain segments that are hydrogen bonded
`together. Such an effect on viscosity is ”tantamount to increasing the number
`and/ or length of polymer molecules. The additive effect would then be
`overtaken and decline as simultaneous chain scission due to thermal degra-
`dation continues.”49
`
`2.3.7 Ultrasonic Depolymerization
`
`Ultrasonication has been used as a conventional method of degradation to
`obtain polymers of lower MW. The generally accepted theory is that ultra-
`sonic depolymerization proceeds by mechanical force. Propagation of acous-
`tic energy causes rapid pressure variation to form small bubbles (cavitation)
`in the liquid. Subsequently, the bubbles collapse with large velocity gradi-
`ents, and this collapse is responsible for the breakage of polymers.50
`Miyazaki et a1. investigated the effect of sonication intensity, temperature,
`HA concentration, coexisting cations, and ionic strength on the depolymer-
`ization of HA in solutions. Size exclusion chromatography with a low-angle
`laser light-scattering photometer was used to measure the size change of
`the HA molecules. The initial depolymerization rate k was found to increase
`with the sonication intensity, but the ultimate depolymerized MW (Mm)
`generally converged to the same value. For example, continuous sonication
`with 55 W depolymerized the HA to a Mlim of approximately 0.1 x 106.
`However, the Mlim almost tripled in the presence of concentrated monovalent
`cation, such as in a solution of 7M LiCl.
`
`Miyazaki et al. also found that the degradation product of HA by ultra-
`sonification had a narrow distribution of MW (MW/Mn slightly above 1). This
`is different from degradation of HA by heat, which broadened the MW
`distribution of HA by random scission. It was demonstrated that the low-
`MW samples with desired, narrow size distribution could be prepared by
`adjusting the sonication intensity or the constitution of the solution}?0
`
`
`
`2.4 Chemistry in Isotopic Labeling
`
`The need for an isotopic labeling technique has been a significant impetus
`behind the research and development of many chemical processes of HA.
`The utility of such labeling is twofold. First, the labeled HA can play a role
`as a tracer in metabolic and phargraccfiinetic studies. Second, the labeled
`age
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`Chemistry
`
`43
`
`HA can serve as a probe for proteins and cell surface receptors that specif-
`ically bind to HA.
`HA has ubiquitous presence in human and animal tissues, and HA is
`constantly metabolized in the body. There are obvious advantages of using
`radiolabeled HA in the study of HA metabolism-"1r52 Radiolabeled exogenous
`HA can be easily distinguished from the endogenous HA of the study
`subjects, and therefore it is not necessary to compare data about exogenous
`HA with an ever-changing baseline of endogenous HA. For the study of
`absorption, distribution, and elimination of exogenous HA, using radiola-
`beled HA is often the method of choice.
`
`There are two major methodologies for radiolabeling HA. One is chemical
`modification, and the other is biosynthesis. Chemical methods involve chem-
`ical reactions of HA with radioactive reagents containing 3H, 1“C, 1751, 11C, and
`so on. Sometimes it requires that the target HA be chemically modified before
`a radioactive moiety can be introduced. Biosynthesis uses cell culture or fer-
`mentation processes with radioactive precursors (3H or 14C) such as glucose,
`acetate, and so forth.53r54 Biosynthetic techniques can provide radioactive HA
`structurally identical to the natural HA. However, the processes are laborious,
`and they do not provide much control of the MW of the HA process.
`Several chemical methods have been reported in the literature.”62 Each
`method has limitations as well as values, depending on the particular
`intended applications.
`
`2.4.1
`
`Exchange Reactions
`
`Tritium gas exchange reaction for tritium labeling"5 in theory should not alter
`the chemical structure of HA. However, it requires several days of exposure
`of HA solutions to tritium gas, during which serious degradation of HA can
`occur. Moreover, much of the tritium labeled to HA is labile, as it is associated
`
`with the hydroxyl groups of HA. It is not practical to completely remove
`the labile tritium, nor is the labeled HA stable under storage. This method
`appears to be applicable mainly for the making of radioactive HA
`oligosaccharides56 with MW less than a few thousand dalton.
`
`2.4.2 N-Acetylation
`
`One of the chemical methods used to prepare a stable, tritium-labeled HA
`was to incorporate tritium as part of the methyl group in the N—acetyl group.
`As a first step, the N-acetylglucosamine moiety needs to be deacetylated. In
`addition to the alkaline deacetylation, mentioned earlier in this chapter,
`deacetylation of HA by hydrazinolysis was also experimented with”,57 Hook
`et a1. adopted the method by Dmitriev, in which HA and other glycosami-
`noglycans were mixed with hydrazine, hydrazine hydrate, and hydrazine
`sulfate, sealed in tubes and heated at 105°C.63 The macromolecular HA was
`
`from rooster comb source. No details were given about the effect of the
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`3 R
`
`
`
`heological Properties onyaluronan
`as Related to Its Structure, Size, and
`
`Concentration
`
`
`
`3.1 Some Basic Concepts of Intrinsic Viscosity
`
`There have been extensive publications on the rheology of hyaluronic acid
`(HA), many of which take a complicated, theoretical approach to the
`subject}—11 The goal of this chapter, however, is to put in perspective some
`basic concepts of the rheology critical to the understanding of the bio-
`physical property of HA and its derivatives, while reducing the mathe-
`matical formula and calculations to the minimum. The study of the
`structure—property relationship has always been an essential part of the
`basic science for any biomaterial, and for HA, the property that stands
`out is its viscosity.
`
`3.1.1
`
`Intrinsic Viscosity — A Measure of Hydrodynamic Volume
`
`The viscosity of a fluid by definition is a measure of its resistance to flow.12
`Perhaps what interests us most from the chemistry standpoint is the intrin-
`sic viscosity (IV) that largely reflects the intrinsic nature of the polymer —
`its molecular weight (MW), primary and secondary structures, and so on.
`IV [1]] is usually measured by a capillary viscometer, and a series of dilu-
`tions of the polymer are allowed to pass through the capillary under
`gravity.
`The ratio t/ta, the efflux time t of the solution over the efflux time to of the
`solvent, is defined as relative viscosity 11,. Specific viscosity is defined as
`115', = n, — 1, and reduced viscosity is given as nwd = nsp/ C, in which C is the
`concentration (g/ ml). The extrapolation of reduced viscosity to zero concen-
`tration gives IV [11]. The unit of IV is milliliters per gram, which has impor-
`tant significance in the study of HA and its derivatives. IV is the measure
`of the hydrodynamic volume of polymers (Fig. 3.1).12
`
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`79
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`80
`
`Practical Aspects of Hyaluronan Based Medical Products
`
`
`
`Concentration of HA10‘4(3 / ml)
`
`FIG U RE 3.1
`
`Intrinsic viscosity [1]] is measured by extrapolating reduced viscosity to zero concentration.
`
`3.1.2 End-to-End Distance Determines Hydrodynamic Volume
`
`The hydrodynamic volume as a macroproperty of polymers is in essence a
`measure of its molecular size.12 One way to describe the molecular size of a
`linear polymer is to use the end-to-end-distance theory.13 According to that
`theory, the average end-to-end distance of the polymer determines the
`hydrodynamic volume of a linear polymer. In other words, the molecular
`size is conceptualized as how much space a linear polymer molecule can
`occupy in the solution, which is dependent on the time-average distance of
`the two ends of the linear polymer chain.
`
`3.1.3 Extended Conformation Increases Molecular Size
`
`A question of considerable interest is what factors determine the end-to-end
`distance of a polymer, One obvious answer is the MW, A higher MW means
`more atoms and therefore longer contour chain length — the sum of the
`lengths of all bonds. The ”random-flight” model is a mathematical tool used
`to calculate the end-to-end distance from the bond length and bond number,
`assuming that the linear polymer chain had complete flexibility.13 The equa-
`tion for such imaginary conditions is:
`
`Distance 2 bond length x bond numbers“2
`
`This random-flight model is in reality far from being a close approximation
`to the reality, because atoms of the polymer molecule are not in absolutely
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`

`Rheological Properties of Hyaluronan
`
`81
`
`random motion (although the term ”random coil” is used in its relative
`sense). All polymer molecules are more or less extended, and therefore the
`actual end-to-end distance is always larger than calculated from the random-
`flight model. It is obvious that an extended conformation of a polymer is
`favorable to the increase of molecular size, and therefore the increase of its
`
`hydrodynamic volume. This is further examined, as follows, from the struc-
`tural characteristics of a molecule.
`
`3.1.4 Structure Features Favoring Extended Conformation
`
`Bond angles of polymer chains are fixed and always larger than 90°. Rota-
`tions of the chain bonds are somewhat restricted, even with highly flexible
`polymers such as polyethylene. The repulsion between adjacent hydrogen
`atoms makes certain more folded conformations very unfavorable.
`The polysaccharide chains are linked by sugar rings. An interesting com-
`parison was made between the IVs of the linear dextran and amylose with
`the same MW (1 million Da).14‘16 Under the aqueous condition, the IV of
`amylose was 159 ml/ g, and that of dextran was 98 1111/ g. The lower hydro-
`dynamic volume of dextran is a result of the additional freedom of rotation
`at C5-C6 in 1,6-linked polysaccharides (Fig. 3.2).17 For polysaccharides in
`which successive residues are separated by only two bonds (such as 1-4
`linkages in amylose), the rotations are more restricted, and less than 5% of
`the conformational space is readily accessible”19
`The functions and properties of polysaccharides, as well as proteins and
`nucleic acids, are all related to their secondary structures. The difference,
`however, is that polysaccharides are generally composed of repeating sac-
`charide units that act cooperatively to form chain packing. This compensates
`for the entropy drive and makes the extended conformation more favorable.
`Chain folding within a confined volume, as seen in globular proteins, is rare
`for polysaccharides.20 The secondary structure has an even more significant
`effect on HA, as will be discussed later.
`
`mo
`
`H
`
`R3
`
`H
`
`HO
`
`CHZOH
`
`0
`
`1
`
`0“
`
`CH20H
`
`4
`HO
`
`0
`
`HO
`R2
`
`0
`
`0H
`
`1
`
`0
`
`5
`
`HO
`HO
`
`0
`
`HO
`
`O~
`
`R4
`
`amylose.1-4 linkage
`
`dextran, 1-6 linkage
`
`MW = 1 million,
`
`IV =159 mllg
`
`MW = 1 million, IV = 98 mllg
`
`FIGURE 3.2
`
`Comparison of hydrodynamic volume of 1 million Da molecular weight amylose and dextran.
`Page 15
`
`© 2006 by Taylor & Francis Group, LLC
`
`Pa