5
`Chemical Modifications, Solid Phase,
`Radio-Chemical and Enzymatic
`Transformations of Hyaluronic Acid
`
`From the chemical point of view, hyaluronan possesses four different types of functional
`groups: acetamide, carboxylic acid, hydroxyl and terminal aldehyde. After deacetylation, a
`free amine could be obtained from an acetamide group. All four functionalities permit
`characteristic chemical reactions. Such a wide variety of possible chemical modifications
`creates a sharp difference between hyaluronic acid and other polysaccharides whose
` reactivity depends mainly upon hydroxyl groups.
`We realize that the description of the overall reactivity and chemical modifications of hya-
`luronan is a dense subject that could fill a separate monograph. However, this chapter will
`focus mainly on the chemical modifications of hyaluronan that lead to cross-linking. Such
`modifications play an important role for the creation of hyaluronic acid with valuable chemical
`and physical properties necessary for biological application of hyaluronan products.
`Historically, hyaluronan was known for the chemical transformation of its hydroxyl
`groups. Only recently, several processes also described transformation of carboxyl groups
`and deacetylated amino groups. Generally, polysaccharides, including hyaluronan, possess
`all chemical reactions characteristic to hydroxyl-containing compounds including
`ethers and ester formation, substitution, elimination, and so on. The reactivity of hyalu-
`ronic acids depends mainly on the functionality of hydroxyl groups. Also, the contri bution
`of the aldehyde group is relatively small. The presence of the free hydroxyl groups provides
`the possibility of structural modifications of the sugar base, which allows direct bio-specific
`modification to be carried out by means of, for example, bi-functional reagents that interact
`simultaneously with two functional groups of neighbouring macromolecules. We consider
`bi-functional reagents as chemical compounds that usually possess two of the same reac-
`tive groups separated by a spacer. Bi-functional reagents are widely used for a covalent
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`Hyaluronic Acid: Preparation, Properties, Application in Biology and Medicine, First Edition.
`Mikhail A. Selyanin, Petr Ya. Boykov and Vladimir N. Khabarov.
`© 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.
`
`ALL 2055
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`122 Hyaluronic Acid
`
`linkage of sterically close fragments of polysaccharide macromolecules. One method of
`bio-specific modification is photo-initiating polysaccharide linkage with an initial intro-
`duction of photo-reaction fragments into its structure.
`This chapter focuses on two subjects related to the field of mechanically stimulated
` reactions: (1) chemical and photochemical cross-linking of HA in aqueous solutions and (2)
`advanced methods of solid-phase polysaccharide modification. From these processes, the
`cross-linked hyaluronic hydrogels acquire a number of valuable properties that significantly
`extend the range of their medical applications.
`
`5.1 Main Characteristics of Cross-Linked Hydrogels
`
`In aqueous solution, hyaluronan forms a gel-like structure as a result of intermolecular
`interaction of linear macromolecules. Colloidal chemistry defines gels as structured sys-
`tems with liquid dispersion media that exhibit mechanical properties more or less similar
`to those of solids. The particles of the dispersed phase are connected to each other in a
`three-dimensional web (which contains dispersion media in its cells and, in the case of
`hydrogels, water) that deprives the system of fluidity. It is obvious that the properties of
`hydrogels are mainly dependent on the strength of bonds and level of reticulation in the
`cross-linked structure. Cross-links in biopolymers can be categorized as either physical
`(formed by electrostatic interactions or hydrogen bonds) or chemical (formed by covalent
`bonds). The physical gels, upon heating, undergo web node decomposition that leads to a
`reduction in the shift modulus. Chemically cross-linked gels are significantly more resist-
`ant to heat but at high temperatures a complete and irreversible destruction of the gel’s
`chemical structure takes place. The level of cross-linkage is determined by the average
`molecular weight of the polymer chain located between cross-links. A cross-link’s density
`directly affects the fundamental properties of the hydrogels, such as degree of swelling,
`mechanical strength and elasticity, permeability and diffusion characteristics [1].
`The hydrogels, formed by hyaluronan as a result of cross-linking, are amphiphilic poly-
`meric substrates able to swell in water and form an insoluble bulky web. The polymeric
`network is in equilibrium with aqueous environment while there is the balance of elastic
`forces of cross-linked polymers with osmotic forces of solution. The chemical composi-
`tions and the molecular weight of the macromolecule fragment between two cross-links
`determine a density of the cross-links that, in turn, influences the swelling and size of gel
`pores [1, 2]. Besides that, the cross-linking characterizes hydrogel as a pseudo-solid com-
`pound, not the solution, thereby giving it viscoelastic properties [3]. Such properties are
`expressed through the physical characteristics of the hydrogels, including swelling level or
`amount of absorbed water. The swelling is directly related to the chemical structure of the
`polymer and is in inverse proportion to the density of the cross-links.
`In 1943, Flory and Rehner were the first to find a connection between the level of cross-
`link density of the polymer and its swelling [4]. In the Flory–Rehner model, the swelling
`level is determined by equilibrium between elastic properties and the forces originated
`from mixing the polymer and solvent. In 1977, Peppas and Merrill modified the theory of
`Flory–Rehner to apply it to the behaviour of the hydrogels [5]. Due to elastic forces, the
`presence of water affects change in chemical potential inside the system [5] and the chemical
`structure affects the swelling. For example, hydrogels with hydrophilic groups, to which
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`Chemical Modifications, Solid Phase, Radio-Chemical and Enzymatic Transformations
`
`123
`
`HA is related, swell more than hydrogels with hydrophobic groups; the latter do not
`increase in volume in the presence of water [6]. The swelling hydrogel very often may
`depend on pH, temperature or other factors [7].
`The limit of swelling may be determined experimentally or calculated theoretically. The
`accurate measurement of swelling limit is useful in the calculations of cross-link density,
`mesh size and the diffusion coefficient. Many natural gels are formed by polyelectrolytes.
`The physical properties of these systems are greatly influenced by an osmotic pressure
`caused by counter-ions associated with the polymeric chains. Free counter-ions significantly
`increase the swelling of the charged gels and affect the elastic modulus. The mechanical
`characteristics of charged hydrogels determine the properties of many biological structures
`such as cartilage, synovial fluid, cornea and striated muscle. In order to measure the swell-
`ing of hydrogels, different experimental methods could be used. An important feature of
`hydrogels is the porosity or the size of the mesh. It is a structural property of the material,
`which is determined as a distance between adjacent cross-links. The study performed with
`polyethylene glycol diacrylate (PEGDA) experimentally established significant changes in
`the porosity upon the changes of the polymer molecular weight and small changes of cell
`sizes at the different concentration [8]. Direct measurements of porosity involve electron
`microscopy or quasi-elastic laser scattering. Indirect methods include mercury porosimetry
`and measurements of high elasticity and maximum swelling [7,9].
`In designing cellular tissue, the diffusion rate of the solubilized compound is important
`in order to determine the rate of release of drugs or transport of nutrients and metabolites.
`The diffusion of nutrients, metabolites and other solubilized compounds depends on many
`factors, including the morphology of the network, the chemical composition of the hydro-
`gel, the water content, the concentration of solubilized compounds and the level of the
`material swelling [7].
`The nature of the cross-links affects the formation of the hydrogel, its shape, size and
`degradation. The formation of cross-links should be monitored for the biomedical applica-
`tions of hydrogels. In this section, three different types of cross-linking in hydrogels are
`described: covalent, ionic and physical interactions [10].
`The appearance of the chemical covalent cross-links can take place during the radical
`polymerization under exposure to high-energy radiation (gamma and electron) [10]. Before
`radical polymerization the polymers are usually modified by adding additional reactive
`groups. For example, acrylate is added to polyethylene glycol (PEG) in order to achieve
`covalent cross-linking [11]. The radical polymerization of acrylate groups can be initiated
`by light, high temperature and redox catalysis [12,13].
`After the cross-linking process is started, it cannot be cancelled or stopped; it is controlled
`only by the initial process conditions. Photopolymerization is the conversion of a liquid
`polymer solution to gel under the action of photosensitizing additives and light [10] and is
`the most ideal method for synthesis of cross-linked hydrogels intended for use in medical
`practice since it allows for the reaction to be carried out with almost 100% efficiency.
`The second type of chemical bonds in hydrogels represents the bonds based on ionic
`interactions. Several natural polysaccharides – for example alginate, a natural polysaccharide
`made from algae, hyaluronan and other charged polymers – form the hydrogel with ionic
`interaction in the presence of bivalent or multivalent cations. The reaction typically
` proceeds at ambient temperature and neutral pH [14]. The ionic interactions are weaker
`than covalent bonds, so such hydrogels undergo rapid degradation in physiological solution
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`124 Hyaluronic Acid
`
`(the media in which they are supposed to be used). One example of a synthetic polymer that
`forms hydrogel with ionic interactions is poly[di(carboxylatephenoxy)phosphazene] [15].
`The weakest type of interaction realized by hydrogen bonding, hydrophobic interac-
`tions and Van der Waals forces also leads to gel structuring. Hydrogen bonds are usually
`stronger than hydrophobic and Van der Waals interactions – their energy values are in the
`range of 10–40 kJ/mol, but they are still an order of magnitude weaker than the ionic and
`covalent bonds. These weak interactions, however, play a central role in the process of
`molecular self-assembly, since the various combinations of these interactions in the
`macromolecules lead to a strong binding. R. Zhang et al. described the molecular self-
`assembly as a set of molecular building blocks that spontaneously form stable, physi-
`cally connected network structures [16]. Despite the weakness of each act of physical
`binding, the multiplicity of such links makes the gel network structures quite stable.
`Thus, the various chemical and physical bonds can participate in the formation of stable
`cross-linked hydrogels.
`
`5.2 Methods of Hyaluronic Acid Cross-Linking
`
`As was mentioned previously, hyaluronic acid possesses four functionalities: acetamide,
` carboxyl, hydroxyl and terminus aldehyde. All are suitable for cross-linkage reactions.
`Depending on the nature of the cross-linkage reagent, a large variety of hyaluronic acid
`materials, starting from the films with low water content up to hydrogels with high water
`content have been synthesized. The majority of the methods of the production of cross-linked
`hyaluronan is related to one of two schemes: (1) a one-stage process with a bi-functional
`reagent able to create cross-linked bridges or (2) a two-stage process in which highly reactive
`HA derivatives are synthesized then followed by second reaction that creates cross-links.
`Different reagents are typically used for hyaluronan cross-linking including diamines,
` aminoaldehydes (obtained from aminoacetals), dialdihydes, butadienesulfones, diepoxides,
`salts of divalent metals and others [17].
`
`5.2.1 Cross-Linking with Carbodiimides
`
`One of the most common reactions of the HA carboxylic group with amino acids and
`diamines is condensation in presence of HOBT in water/DMSO [18]. It is known that one
`of the best condensation methods of carboxylic and amino functions is the reaction in the
`presence of DCC (dicyclohexylcarbodiamide). One of the first studies of the condensation
`with DCC was conducted in 1991 [19]. A similar method was used for reaction with differ-
`ent amines [20]. Unfortunately, DCC required the reaction in non-aqueous conditions.
`However, there is a possibility to perform cross-linking with EDC – water soluble analogue
`of DCC [21]. A similar process, which mentioned condensation with EDC and resulted in
`cross-linked hydrogels is described in [22,23].
`General methods for cross-linking of biopolymers such as hydroxyethylcellulose (HEC),
`carboxymethylcellulose sodium salt (CMC Na) and hyaluronic acid (HA) using water
` soluble carbodiamide are summarized in a review published in 2005 [24]. The interesting
`invention described the synthesis of water-insoluble derivative of hyaluronic acid cross-
`linked with biscarbodiimide [25].
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`Chemical Modifications, Solid Phase, Radio-Chemical and Enzymatic Transformations
`
`125
`
`O
`
`HA C O
`
`HA
`
`(A)
`(B)
`
`X–
`
`R R
`
`H
`+
`C N
`C N
`
`R
`R
`
`N
`N
`
`OH
`
`+
`
`HA
`
`OH
`
`CO
`
`HA
`
`A –EDC or analogues
`B –DDC or analogues
`
`Figure 5.1 Hyaluronic acid cross-linking with carbodiimide
`
`R2
`
`NH
`
`O
`
`R3O
`
`R3O
`
`R4
`
`O
`
`O
`
`O O
`
`N
`
`OR3
`
`O
`
`COR
`
`H3COC
`
`NH
`
`O
`
`O
`
`O
`
`O
`
`OR3
`
`R4
`
`R1
`
`R3O
`
`OR3
`
`Figure 5.2 Cross-linked hyaluronic acid with amide bond after reaction with carbodiamide
`using the Ugi approach
`
`One of the latest studies investigates the role of the solvent in carbodiimide cross-linking
`of hyaluronic acid. The cross-linked products were intended for use in ophthalmology. The
`conclusion was made that after the EDC treatment in the presence of an acetone/water
`mixture (85:15, v/v) the HA hydrogel membranes have the lowest equilibrium water con-
`tent, the highest stress at break and the greatest resistance to hyaluronidase digestion.
`Irrespectively of the solvent composition (in the range of 70–95%), the cross-linked HA
`hydrogel membranes are compatible with human RPE cell lines without causing toxicity
`and inflammation [26].
`An interesting method of connecting two hyaluronic acid fragments through carboxylic
`and primary amine functions using the Ugi approach is described in the patent [27]. In the
`method the primary amine was generated by deacylation, then two molecules were allowed
`to react in the presence of formaldehyde and cyclohexylisocyanide (Figure 5.2). The Ugi
`reaction allowed scientists to make cross-linked HA with an N-substituted amide bond, in
`which carboxylic and amino functions came from the different HA molecules.
`Another possible way to synthesize cross-linked HA is by using bi-functional reagents.
`The cross-linked HA product is synthesized after reaction of HA with dihydrazide in the
`presence of HOBT and carbodiimide (Figure 5.3) [28]. In this reaction only the carboxylic
`functions from both HA molecules were used for cross-linking.
`Another example of the synthesis of cross-linked HA with a dihydrazide bridge is
`described in [29]. For a formation of the bond between hyaluronan and primary amine,
`carbodiimide and N-hydroxysulfosuccinimide were used. The authors synthesized many
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`126 Hyaluronic Acid
`
`O
`
`C HA
`
`HN
`
`HN
`
`O
`
`C
`
`O
`
`C
`
`CH2
`
`n
`
`HN
`
`HN
`
`NH2
`HA
`
`O
`
`C
`
`HN
`
`O
`Cn
`
`C
`
`CH2
`
`HN
`
`H2N
`
`O
`2HA C
`
`OH
`
`Figure 5.3 Cross-linking of HA with dihydrazide
`
`First step
`
`Second step
`
`HA
`
`HA intermediate
`
`Double crosslinked
`HA network
`
`Figure 5.4 The process of double cross-linking of HA [21]. Reproduced with permission from
`[21]. Copyright © 1997 John Wiley & Sons, Inc.
`
`2HA CH2 OH
`
`CH2O
`
`HA CH2
`
`O
`
`CH2
`
`O
`
`CH2 HA
`
`Figure 5.5 Cross-linking of HA with formaldehyde
`
`polyvalent hydroxide reagents (2–6 hydrazydes per reagent) for the use in the reactions of
`HA cross-linking by carbodiimides.
`In order to carry out cross-linking, Zhao et al. developed a two-stage method where syn-
`thetic polymer polyvinyl alcohol was used at the first stage, followed by ionic biopolymer
`sodium alginate in combination with hyaluronate (Figure 5.4) in the second stage [30]. This
`method allowed for the polymer network to be obtained with increased biostability.
`
`5.2.2 Cross-Linking with Aldehydes
`
`Formaldehyde and glutaraldehyde have long been used to cross-link proteins for tissue
`conservation. Formaldehyde is used for synthesis of the several cross-linked HA products
`(Figure 5.5). Cross-linking with glutaraldehyde leads to the materials with a high resist-
`ance to biodegradation. Tomihata and Ikade studied the reaction with glutaraldehyde in the
`acidic aqueous solution with acetone with the purpose of obtaining hyaluronic films with
`restricted swelling [31,32]. Through a comparison of the HA gels cross-linked with gluta-
`raldehyde and carbodiimide, it was found that treatment with carbodiimide leads to final
`products with a larger amount of cross-link bonds. The obtained materials are used for
`skeletons of the tissues [33].
`
`5.2.3 Cross-Linking with Divinylsulfone
`
`The stable hydrogels of HA can be obtained by cross-linking of HA with divinylsulfone.
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`Chemical Modifications, Solid Phase, Radio-Chemical and Enzymatic Transformations
`
`127
`
`CH2
`
`CH2 O
`
`CH2
`
`HA
`
`O S
`
`O
`
`HA CH2
`
`O
`
`CH2 CH2
`
`CH
`
`CH2
`
`O S
`
`O
`
`CH2 CH
`
`2HA CH2 OH
`
`Figure 5.6 HA cross-linking with divinylsulfone
`
`O
`
`Ca2+
`
`2HA C OH
`
`HA
`
`O
`
`C
`
`O
`+Ca+ –O C HA
`
`O–
`
`Figure 5.7 HA cross-linking with bivalent metal ions
`
`In alkaline media divinylsulfone or butadienesulfone, sulfonyl ether linkage is formed
`between hydroxyl groups of HA. Depending on the reaction conditions, the reaction prod-
`ucts could have different consistencies from the soft gels up to solid films (the same method
`could be used for formation the membranes and hollow pipe). Such products, used as
`microimplants, are allowed to stay in the body for a long time, especially in the places
`where implants are not affected by mechanical forces.
`
`5.2.4 Cross-Linking by the Ions of Polyvalent Metals
`
`Adding polyvalent metal salts into the solution of hyaluronan leads to physical cross-linking
`of polysaccharides due to the formation of ionic bonds. For example, divalent metals such
`as calcium, zinc, copper and so on, form cross-linked HA salts [34]. However, such ionic
`binding with polyvalent metals (Figure  5.7) is significantly weaker compared to strong
`chemical covalent bonds. Stability of such ionic binding depends on different factors such
`as pH, media, ionic strength and temperature. So, when such hyaluronic acid salts are
`used as biomedical materials, for intradermal injection in mesotherapy for example, their
`presence in derma has a short life span and it is difficult to control in order to assure the
`necessary physiological action of HA hydrogels on the organism [35].
`
`5.2.5 Cross-Linking with Epoxides
`
`Almost 40 years ago, Laurent et al. obtained cross-linked hyaluronan hydrogels using
`diglycidyl ether of polyetheleneglycol [36]. Other researchers extended the possibilities of
`diepoxyde chemistry using ethelene glycol diglycidyl ether as a bi-functional reagent and
`glycidyl ether of polyglycerol as a trifunctional reagent. The cross-linking method by
`epoxides is described in [37]. The detailed study of the reaction mechanism showed that at
`the high pH diepoxides form ester bonds involved carboxylic groups, while at low pH ether
`bonds are formed between hydroxyl groups.
`The cross-linking method with diepoxide and epoxide oligomers has great potential
`because the products, especially in combination with ether links, possess relatively high
`resistance to hydrolytic decomposition. It is related to the reaction products of diepoxide of
`bis-alcohols and ethylene glycol oligomers including bis-ethylene glycol [38]. One of the
`most common methods for HA cross-linking is using diepoxide of bis-alcohols, particularly
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`128 Hyaluronic Acid
`
`HA
`
`O C
`
`O
`
`HA C
`
`O
`
`OH
`CH2 CH
`
`OH
`
`R
`
`CH
`
`CH2
`
`O
`
`O
`
`2HA C OH
`
`CH2 CH
`O
`
`R CH
`
`CH2
`
`O
`
`Figure 5.8 Cross-linking of HA with BDDE at the carboxylic group (esterification)
`
`2HA OH
`
`CH
`
`R
`
`CH2
`O
`
`CH CH2
`O
`
`pH < 7
`
`OH
`
`OH
`
`HA
`
`O CH2 CH
`
`R
`
`CH CH2 O HA
`
`R = CH2 CH2
`
`O
`
`CH2 CH2
`
`O
`
`CH2 CH2
`
`O
`
`CH2 CH2
`
`DEG
`
`R = CH2 CH2
`
`O
`
`CH2 CH2
`
`CH2
`
`CH2
`
`O CH2 CH2
`
`BDDE
`
`Figure 5.9 Cross-linking of HA with BDDE at the hydroxylic group (etherification)
`
`1,4-butandioldiglycidyl ether (BDDE) in alkaline media [39]. However, this method has
`several deficiencies:
`
` ● Usage of reagent excess, which makes it difficult to predict the level of cross-linking and
`purification of the final product;
` ● The reaction takes place using the carboxylic groups, which leads to products which are
`less stable for hydrolysis and easy degraded in the body (Figure 5.8).
`
`Another HA cross-linking method with BDDE was described by De Belder and Malson [40].
`The reaction of HA with BDDE could be carried out in acidic media, which was created by
`hydrochloric acid. However, it was proved that in acidic media the cross-linking reaction
`takes place using the carboxylic group as well. The comprehensive study of the BDDE cross-
`linking in different conditions was performed by Schante et al. [41]. It was finally proved that
`contrary to early data, the reaction in acidic conditions leads to the cross-linking at carboxylic
`groups (esterification). The cross-linking in alkaline conditions (pH around 10) takes place at
`hydroxylic groups (esterification), which leads to more stable products (Figure 5.9).
`It is important to mention that cross-linking agents such as carbodiimes, aldehydes and
`epoxides, are toxic compounds. Their presence in the final medicinal product, even in small
`amounts is absolutely unacceptable. Purification of the cross-linked hyaluronate gel from
`technological impurities is a very difficult process that leads to considerable increase in the
`processing cost of goods. That is why it is important to develop technologically ‘clean’
`cross-linkage processes based on the physical methods of the chemical reactions, such as
`solid-state modification and photo-cross-linking.
`
`5.2.6 Photo-Cross-Linking
`
`A formation of cross-linked hyaluronan could be achieved by UV-exposure. This is how
`two covalently linked hydrogels were produced and used in tissue engineering [42]. A
`modification of hyaluronan and other similar polymers by UV exposure with added photo-
`active molecules increases the biochemical functionality and mechanical stability of the
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`Chemical Modifications, Solid Phase, Radio-Chemical and Enzymatic Transformations
`
`129
`
`hydrogels. The molecules of polyethelene glycol acrylate (PEGA) undertook radical pho-
`topolymerization in order to form cross-linked hydrogels [43].
`In order to initiate photo-radical reaction of the chemical cross-linking in HA, its
` functional groups undergo structural modifications. Leach et al. carried out the reaction
`between the methacrylic esters and hyaluronate [44]. Shu and Prestwich [45] successfully
`synthesized HA thioates. Raeber et al. were the first to modify PEGA with the fibronectin
`derived integrin-binding peptide Ac-GCGYGRGDSPG in order to increase cell adhesion
`and tissue growth stimulation [46]. Similar transformations were carried out with hyaluro-
`nan for subsequent bone engineering applications, namely to enhance cartilage repair [45].
`It is important to point out that it is possible to carry out photochemical transformations
`in mild conditions in order to maintain the activity of the biological molecules or encapsu-
`lated cells. For such purposes the photo-transformation of HA is carried out in the presence
`of several compounds, such as cinnamic acid derivatives, coumarin, thymine, methacrylic
`anhydride, glycidyl methacrylate and styrene. Figure 5.10 shows how methacrylic esters
`undergo photopolymerization in the presence of HA to form a grafted polymer [46].
`The advantage of such method is that the product of HA photo reactive derivatization is
`a water-soluble compound. Before the process of photo-solidifying starts, when the three-
`dimensional web has not yet formed, unreactive toxic compounds with low molecular
`weight could be easily removed from the reaction zone.
`Photo solidifying is thus the last step of a two-stage method of gel cross-linking that takes
`place instead of the reaction with chemical reagents. At the same time, the first stage includes
`two operations – the introduction of spacer followed by a photoactive group [35]. As a
`result, UV exposure permits cross-linked hydrogels to be obtained, which are impurity-free
`and ready to use. At the same time, this method, as all processes are carried out in the liquid
`phase, does not ensure equal density of cross-linkage in the whole material volume. This is
`related to the fact that all synthetic polymers are dispersed by the molecular weight and, in
`other words, represent a mixture of macromolecules of different sizes.
`The natural biopolymers, to which HA belongs, are not polydispersed polymers due to the
`matrix nature of their synthesis. The nature of the biochemical synthesis is determined by the
`matrix; the enzyme upon which the biopolymer is synthesized. Nevertheless, during biopoly-
`mer extraction and purification processes they degrade in one way or another. For example,
`polygalactomannan, different types of cellulose (wood or cotton), chitosan and hyaluronan
`are isolated as a wide range of the relatively narrow dispersed macromolecule fractions.
`The purification and isolation of the final product of the biochemical synthesis, as a rule,
`always provides a multimodal set of narrow fractions (multimodal type distribution is a mode
`in which more than one peak appears on the graph of the molecular weight distribution).
`This leads to the fact that during the binding of a polydisperse system under mild conditions
`
`O
`C
`
`CH3
`C
`CH2
`
`2H
`
`O
`
`2HA
`
`CH2
`
`OH
`
`2HA
`
`CH2
`
`O
`
`O
`CH3
`C C
`CH2
`
`Photoreagent
`hv
`
`HA
`
`CH2
`
`O
`
`O
`C
`
`CH3
`CH
`
`CH2
`
`CH2
`
`CH3
`CH
`
`O
`C
`
`O
`
`CH2
`
`HA
`
`Figure 5.10 Cross-linking of HA by photopolymerization
`
`Copyright © 2015. John Wiley & Sons, Incorporated. All rights reserved.
`
`Khabarov, V. N., et al. Hyaluronic Acid : Production, Properties, Application in Biology and Medicine, edited by Felix Polyak, John Wiley &
` Sons, Incorporated, 2015. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/sandiego/detail.action?docID=1895546.
`Created from sandiego on 2020-06-26 07:21:27.
`
`

`

`130 Hyaluronic Acid
`
`in the aqueous media, the high molecular mass fractions are cross-linked faster than low
`molecular mass fractions. The result is the obvious problem of heterogeneity of the gel, which
`is especially noticeable at high degrees of cross-linking. These disadvantages can be avoided
`by using solid-phase modification process polysaccharides (solid-state reactive blending
`(SSRB) [47].
`
`5.2.7 Solid-State Cross-Linking under High Pressure and Shear Deformation
`(Solid-State Reactive Blending: SSRB)
`
`Since the 1970s, systematic studies of the effects of high pressure and shear stress on solid
`organic compounds have been conducted. The main instrument used in the studies was the
`Bridgman anvils, the device invented by Nobel Prize winner P.W. Bridgman [48] for studying
`the detonation properties organic explosives. During that period a number of observations on
`the evolution of certain organic substances under high (1.5 GPa) pressure and shear deforma-
`tion were published. More than 40 years ago, Enikolopov published the first report regarding
`the polymerization of several solid-state vinyl monomers under the effect of high pressure
`and shear stress. Since then the conversions of hundreds of organic compounds and different
`types of chemical reactions taking place in these conditions have been studied [49]. Many
`chemical reactions involving both low molecular mass substances and polymers had been
`carried out by way of direct and reversed etherification and amidation. These reactions
`involve epoxides, various types of rearrangements, polymerization and polycondensation
`and modification of polymers of different structures. The rules identified in these studies
`demonstrate a critical role of deformation in determination of the conversion degree and
`existence of critical pressure, below which the reaction does not occur at all degrees of
`deformation.
`The identification of the active states during the different conditions of the mechanical
`action permits establishment of possibilities to carry out chemical processes and make a prog-
`nosis for how to use them effectively. Deformation of solid materials, regardless of their chem-
`ical nature, is accompanied by deep disordering of the solid compound through the creation of
`nano-sized structures and a large amount of active centres (electron- and vibration-exited
`bonds, electrons and ions stabilized in the traps, low-coordinated atoms in the dislocation
`nucleus and other structural defects, meta-stable atoms etc.). This is w