i
`
`Chemical modification of
`hyaluronic acid for drug delivery,
`biomaterials and biochemical
`probes
`
`Glenn D. Prestwich* tri, Dale M. Marecak*-j-, James F. Marecekf,
`'Coen P.Vercruysse *t and Michael R. Ziebell *§
`*Department of Medicinal Chemistry. 308 Skaggs Hall, College of Pharmacy,
`The University of Utah, Salt Lake City, Utah 84112, U.S.A., -[Department of Chemistry,
`University at Stony Brook, Stony Brook, New York 11794-3400, U.S.A., fDepartment
`of Biochemistry & Cell Biology, University at Stony Brook, Stony Brook. New York
`11794 -3400, U.S.A, and §Department of Physiology & Biophysics, University at Stony
`Brook, Stony Brook, New York 11794 -3400. U.S.A.
`
`Introduction
`
`Hyaluronic acid (HA) is a linear polysaccharide consisting of alternating 1,4-
`linked units of 1,3- linked glucuronic acid and N- acetylglucosamine (Figure 1),
`and is one of several glycosaminoglycan components of the extracellular matrix
`(ECM) of connective tissue [1 ]. Its remarkable viscoelastic properties account for
`its importance in joint lubrication [2] and its complete lack of immunogenicity
`makes it an ideal building block for biomaterials needed for tissue engineering
`[3,4] and drug delivery systems [5,6]. Sodium hyaluronate, the predominant form
`at physiological pH, and HA are collectively referred to as hyaluronan; in this
`paper, the chemical modifications always begin with the carboxylic acid form, and
`we use the abbreviation `HA'. The three -dimensional structures adopted by HA
`and HA oligosaccharides in solution [7,8] show extensive intramolecular
`hydrogen bonding that restricts the conformational flexibility of the polymer
`chains and induces distinctive secondary (helical) and tertiary (coiled coil)
`interactions.
`In this chapter, we will first summarize background information on (1)
`key HA- protein interactions important in cell biology, (2) the role of HA in
`cellular signalling, (3) the biomedical applications of HA, (4) biodegradable
`polymer scaffolds and (5) chemical modification of HA, leading to the discovery
`of the hydrazide modification method. Second, we present an overview of our
`current work on (1) new cross- linkers, (2) optimization of HA hydrogel
`formation, (3) hydrogel degradation by hyaluronidase (HAse), (4) use of
`chemically modified hydrogels for tissue engineering, (5) synthesis and
`applications of functionalized HA probes in cell biology and (6) structural studies
`of HA -HA binding domain interactions.
`
`I To whom correspondence should be addressed (University of Utah).
`
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`43
`
`ALL 2200 FF
`ALL 2200 FF
`PROLLENIUM V. ALLERGAN
`PROLLENIUM V. ALLERGAN
`IPR2019-01505, et al.
`IPR2019-01505, et al.
`
`

`

`44
`
`G.D. Prestwich et al.
`
`Figure 1
`
`CH3
`
`CH3
`
`dl°oIn
`
`CH3
`
`Structure of hyaluronic acid, showing three disaccharide repeat units
`
`HA- protein interactions
`Hyaladherins [9], including the link proteins [10], aggrecan, versican and other
`HA binding proteins, are essential in tissue remodelling processes [11].
`HA- protein interactions are important in cell adhesion, growth and migration
`[9,12,13], stimulation of cell motility and promotion of focal adhesion turnover
`[14], in cartilage [15], in inflammation and wound healing [16] and in cancer
`[17,18]. Many of the molecular details of the interactions of HA with HA binding
`domains (HABDs) of hyaladherins have been elucidated. Indeed, a solution
`structure of the link module, a hyaluronan- binding domain involved in ECM
`stability and cell migration, has recently been published [19]. However, HA is not
`simply `extracellular glue'; the critical importance of HA receptors in the
`regulation of intracellular signalling to the cytoskeleton has been recently
`reviewed [20], and key elements are described below.
`
`Role of HA and HA receptors in metastasis
`Extracellular HA acts as a signalling molecule by activating regulatory kinase
`pathways important for cell cycle progression and movement [21,14,20]. Two
`types of cellular HA receptors have confirmed roles in signalling: (i) CD44, a
`family of glycoproteins originally associated with lymphocyte activation, and (ii)
`RHAMM (receptor for hyaluronan mediated motility), originally identified from
`transformed fibroblasts. CD44 isoforms found on tumour cells bind HA with
`higher affinity and promote cell migration [22]. Turley's group found that
`RHAMM overexpression is itself transforming and regulates events downstream
`from H -ras [23,14]. Moreover, RHAMM plays a role in the cell cycle, inducing
`mitotic arrest by suppression of cdc2 and cyclin bl production [24]. In cancer, HA
`also has effects on angiogenesis and on an increased HA production in and around
`tumours [25].
`While the predicted secondary (and tertiary) structures of RHAMM and
`CD44 are different, both share the HA binding motif common to all known HA
`binding proteins: BX7B, where B is His, Arg or Lys [26]. The importance of these
`two domains for HA binding activity has been determined by deletion mutants
`and synthetic peptide competition experiments. HA-CD44 complexes mediate
`in CD44 was
`lymphocyte -endothelial cell adhesion [27]. A critical Argo'
`identified by mutagenesis [28]. A series of Lys substitutions in RHAMM
`confirmed their importance in the mediation of metastatic transformation induced
`by H -ras [14]. The role of CD44 and RHAMM in determining whether
`transformed cells become aggressive and metastatic has been extensively
`investigated [29,30,22,31 -37]. The importance of HA- receptor interactions in
`
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`
`

`

`Chemically modified hyaluronic acid
`
`45
`
`cancer biology has propelled the development of biophysical and biochemical
`probes in our laboratories, as well as structural studies of RHAMM polypeptides.
`This work will be summarized below.
`The size of the HA oligosaccharide itself is important. Revascularization
`during wound healing is inhibited by high- molecular -mass HA but accelerated by
`shorter HA oligosaccharides [38]. The HA-hexamer blocks HA binding to
`HABD in plasma membrane and HA- decasaccharide is necessary for inhibition of
`the interaction of HA with link protein [39]. Indeed, addition of HA or HA
`oligosaccharides to human articular cartilage explants and monitoring aggrecan
`and proteoglycan synthesis in vitro suggested that chondrocytes may have two
`HABDs, one for uptake of HA for catabolism and one related to hyaluronectin
`[40].
`
`Biomedical applications of HA derivatives
`Both naturally occurring and chemically modified HA have found use in a broad
`range of biomedical applications, including ophthalmic surgery [41] and the
`treatment of arthritis [5,42,43], in particular via the technique known as viscosup-
`plementation [44,45]. A wide variety of uses are described elsewhere in this
`volume, but several examples are described here to place the present chemical
`modification work in context. The chemical derivatization of HA by our labora-
`tories and others provides an opportunity to develop materials for new
`applications that exploit some of the viscoelastic properties of this high -
`molecular -mass polymers. For example, controlled release of pharmacologically
`active compounds such as prednisolone [46,47] for use in eye surgery,
`moisturizing agents, swellable gels, adhesion management aids, cell encapsulation
`matrices, hydrophilic coatings of plastics [48], mammary implants [49] and certain
`aspects of wound treatment [41] are some of the applications envisioned for these
`new materials. Native sodium HA was shown to serve as a template for nerve
`regeneration [50]. HA is suitable for all of these applications since it is bioerodable
`and compatible with systemic functions. Insoluble HA derivatives that retain
`significant bioerodability [51,52] have been used as membranes for the culture of
`keratinocytes for transfer to human wounds [53]. Other chapters in this volume
`will provide an expanded repertoire of the specific uses of HA in human medicine.
`New HA hydrogels can in principle be fashioned into prosthetic implants capable
`of drug delivery [54], into porous microspheres for culturing cells [55,56] and into
`gels for bridging gaps in bone or cartilage during cell regrowth [57 -59].
`
`Biodegradable polymers
`Biodegradable polymer scaffolds based on polyglycolic acid, polylactic acid and
`related co- polymers have been investigated recently as substrates for tissue
`engineering [3], and important advances in neocartilage regeneration [60] and joint
`resurfacing have been described. A high -molecular -mass viscous HA material has
`also been used alone or in gel -like formulations with decalcified bone matrix
`[57,58]. Despite promising results, in these cases, the realization of a shape -
`retaining implant was not achieved. As a result, the need for new biomaterials [4]
`for tissue engineering and for drug delivery [6] continues to drive the
`development of new technologies.
`
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`

`

`46
`
`G.D. Prestwich et al.
`
`Biodegradable hydrogels, extended supermolecules prepared by cross-
`linking macromolecules into a polymer network, have become the subject of
`intense study as carriers for controlled release drug delivery systems [61,62].
`Hydrogels are generally biocompatible, because of low interfacial tension and
`advantageous physical properties for cell adhesion and protein adsorption. The
`major disadvantage is low mechanical strength. New hydrogels based on collagen
`and other naturally occurring components of the ECM have begun to address this
`issue [63,64]. Uses of HA and of HA gels for drug delivery have been reviewed
`[65,66]. Nonetheless, there are very few examples of biocompatible hydrogels
`with well- characterized functional linkers on to which drugs may be appended
`[67]. Thus, improvements in our ability to introduce chemical functionality for
`peptide or drug attachment, in addition to producing a biocompatible,
`bioerodable hydrogel, should provide access to a unique new set of biomaterials.
`
`Chemical modification of HA
`As detailed below, HA can be chemically modified at hydroxy groups of the
`glucuronic acid or N- acetylglucosamine units, the carboxylic acid of the
`glucuronate units, or the reducing end of the polysaccharide chain. Since the
`chemical reactions described herein require the protonated carboxylic acid and are
`conducted between pH 2.0 and 5.0, we will refer throughout to hyaluronic acid,
`or simply HA. The resulting materials used in a biological context would be
`correctly called chemically modified hyaluronan derivatives, referring to a
`mixture of protonated and sodium and /or potassium salts present in physiological
`systems. In this chapter, we will focus on controlled modifications of the
`carboxylic acid under mild aqueous conditions, using water -soluble carbodiimide
`chemistry.
`
`Figure 2
`
`R' N=C=N R2
`
`HA - CO2H
`
`O
`
`HN'R"
`fa.- H+
`R2(1)
`N'
`
`O
`
`HA
`
`R3NH2
`
`O to N migration
`
`nucleophilic addition
`
`/\ R1(2)
`
`N'
`
`HA
`
`O
`
`LR2(1)
`N'
`H
`N -acyl urea adduct
`
`+
`
`HA
`
`NHR3
`
`amide adduct
`
`urea byproduct
`
`Attempted chemical modification of HA with carbodiimides (R' N= C =NR2)
`and primary amines (R3NH2) leads to formation of N -acyl ureas by
`rearrangement of the intermediate 0 -acyl ureas
`The amide and urea by- product are only minor products.
`
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`
`

`

`7
`
`Chemically modified hyaluronic acid
`
`47
`
`Figure 3
`
`Hydrophobic
`
`o
`
`N
`
`Cf3
`
`}
`
`Protected, tethered
`amines
`
`Cross -linkers
`
`}
`
`/
`
`N I
`
`N=C=N
`
`I Designed
`
`Carbodiimides I
`
`IN
`
`N-C-N
`
`/N /N-C=N
`
`N
`
`I
`
`/N\.,.."../
`
`N=C=N
`
`IDesigned Biscarbodiimides
`
`I
`
`/N,,,,,,.......,/N=C=N
`
`I
`
`/N,.,.../...,,/N=C=N
`
`I
`
`Designed monofunctional (top) and bisfunctional (bottom) carbodiimides
`The compounds were synthesized and used to prepare N-acyl urea derivatives and N-acyl urea cross -
`
`linked derivatives of HA.
`
`Carbodiimide modification of glycosaminoglycans has three decades of
`history; unfortunately, for most materials the chemical structures were not well -
`characterized. We initiated our studies of the modification of HA with ethyl(N,N-
`dimethylaminopropyl )carbodiimide (EDCI) and a variety of `designer' carbodi-
`imides in 1986. The production of glucuronamides requires the activation of the
`carboxylic group, which can be accomplished using a water -soluble carbodiimide
`such as EDCI as the condensing agent. The first report of an HA- glycine methyl
`ester derivative [68] could not be repeated in our laboratories [69]. We found that
`the 0-acyl urea activated complex formed between EDCI and high- molecular-
`mass HA (2 MDa) at pH 4.75 did not give the expected intermolecular coupling
`with added diamines or other primary amine -containing nucleophiles (Figure 2).
`Instead, the O -acyl ureas preferentially rearranged to N -acyl ureas rather than
`undergoing coupling to added amine- containing reagents [69]. This was readily
`seen by a characteristic quartet and triplet pattern for the N -ethyl urea moiety in
`the NMR spectrum of these derivatives. Partially degraded HA (60 kDa) also
`followed this reaction pathway. We nonetheless took advantage of this
`observation to prepare lipophilic, aromatic and functionally reactive derivatives of
`HA based on the robust covalent N -acyl urea linkage formed with customized
`carbodiimides (Figure 3, top) [69]. Biscarbodiimides offered the possibility of
`cross -linking HA to form stable gels, and both aliphatic and aromatic biscarbodi-
`
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`
`

`

`48
`
`G.D. Prestwich et al.
`
`imides were prepared (Figure 3, bottom) and shown to give swellable, cross -
`linked products [69,70].
`The unambiguous characterization of the HA N -acyl ureas, as well as
`elucidation of the mechanism of formation of these products, was accomplished
`using isotopically labelled EDCI in conjunction with cross -polarization magic -
`angle spinning (CP -MAS) 13C- and 15N -NMR [71]. Thus, the [2 -'3C] and [1-
`15N]EDCI isotopomers were prepared separately from [2- t3C]ethyl isothio-
`cyanate and 15N- ethylamine, respectively. Using 2 MDa HA at 4 mg /ml with
`sufficient labelled EDCI to obtain a theoretical maximum of 25% modification of
`the available glucuronic acid residues, the 13C- CP -MAS of the product showed a
`new peak at 156 p.p.m. for the N -acyl urea with the expected intensity for 25%
`modification. The chemical shift did not distinguish between two isomeric
`products that could arise from the rearrangement of the EDCI -HA adduct. The
`15N- CP -MAS showed a minor peak at 133 p.p.m. for an unprotonated 15N and a
`major peak at 83 p.p.m. for a protonated nitrogen. These data suggested that the
`O -acyl urea to N -acyl urea conversion was intramolecularly catalysed, as shown
`in the proposed mechanism (Figure 4) [71].
`Meanwhile, many of the other chemical derivatives of HA that involve
`reaction of the hydroxy- and carboxy -functionalities have produced cross -linked
`materials. The preparation of these materials is described in detail by P. Band in
`Chapter 6. For example, HA can be derivatized through the hydroxy group using
`divinyl sulphone cross -linking on the primary hydroxy groups of HA at high pH
`[72]. The hylans [73,74], formaldehyde- cross -linked HA derivatives [75], show
`enhanced rheological properties and longer residence times in tissue relative to
`native HA, but the processing precludes attachment of chemically reactive linkers
`for drug attachment. Alternatively, HA can be partially oxidized with periodate
`and then reductively coupled to Type I collagen [76] to give an artificial ECM, as
`
`\N/
`
`O
`C
`
`HA N
`p 15N
`
`IH
`
`,3
`
`/
`
`C,N
`O,i
`SN
`C` 'C
`
`HA
`
`..151i
`
`H+
`
`Top, structures of 13C -EDCI and 15N-EDCI. Bottom, intramolecular
`catalysis of O- to N -acyl migration during reaction of HA with isotopically
`labelled EDCI
`The regioisomer shown corresponds to an acyl migration of the glucuronate to the more substituted
`nitrogen. The preferred product has a protonated '5N -ethyl and a 13C4abelled glucuronate (arrow), and
`
`was proposed to arise by intramolecular catalysis.
`
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`
`Figure 4
`
`

`

`Chemically modified hyaluronic acid
`
`49
`
`described above [63]. Non -cross -linked ester derivatives have also been
`developed. Fidia Inc. (Italy) employs a method to convert virtually every
`carboxylate group of the glucuronic acid moieties of the HA molecule into an
`ester (e.g. ethyl, benzyl), using the tetra(n -butylammonium) salts of HA dissolved
`in dimethylformamide [77]. This modification makes the HA molecule lipophilic
`[78], and the products have been exploited in gauze mats and microspheres with
`or without ester-linkage tethered drugs [52,51,72,79].
`Insoluble HA derivatives, such as the N -acyl urea cross -linked materials
`[70], have proven useful in prevention of surgical adhesions. Similarly, Genzyme
`has received patents (U.S. No. 4937270 and U.S. No. 5017229) for modifications
`of HA for use in prevention of surgical adhesions [80] and as a drug delivery
`system. Genzyme's modification is to treat HA (or HA- carboxymethylcellulose
`mixtures) with EDCI in the presence of an amino acid to give a water -insoluble
`biocompatible material; the spectroscopic characterization of these products has
`not been presented. Genzyme's Seprafilm ®, a carboxymethylcellulose -HA
`bioresorbable membrane used to prevent surgical adhesions, has received FDA
`approval and is now being marketed. Clinical or preclinical trials from both
`industrial and academic research laboratories with other modified HAs include
`HA- liposomes [81], HA- anti- cancer drugs [82] and HA- anti- inflammatories
`[83,84].
`
`Recently, the problem of obtaining well -characterized, functionalized
`derivatives of HA oligosaccharides was advanced significantly by the discovery
`that dihydrazides could give hydrazide- modified HA for further introduction of
`drug molecules or intermolecular cross -links to produce hydrogels [67,85]. The
`hydrazide- derivatized HA technology exhibited a number of important
`advantages. First, preparation of HA derivatives under mild, aqueous conditions
`preserves the structural integrity and molecular size range of HA. Second,
`homogeneous, modified HA materials can be obtained by removal of all side
`products and unreacted reagents using dialysis, precipitation and /or ultrafil-
`tration. Third, all new HA derivatives based on the bishydrazide technology are
`chemically well- characterized. Fourth, the mechanical and physicochemical
`properties can be engineered to fit a variety of applications, including modifi-
`cation of the molecular mass and the extent of intra- and interchain interactions
`through controlled cross -linking. Fifth, a wide selection of mono -, bis- and
`polyhydrazide linkers can be used for gel production. These reagents may be
`either hydrophobic or hydrophilic and may be chemically robust or readily
`cleavable in vivo. Sixth, the ability to covalently attach proteins, short peptides,
`anti -cancer agents, anti -inflammatory or anti -infective drugs, or cell adhesion and
`growth -promoting ligands to the HA hydrogel provides the potential for drug
`delivery. Finally, HA hydrogels can be incorporated into other biopolymer
`matrices, including the preparation of co- cross -linked biomaterials. The
`remainder of the chapter now deals with the initial development of the hydrazide
`modification strategy and its application to a variety of new, medically useful
`derivatives and biochemical probes.
`
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`
`

`

`SO
`
`G.D. Prestwich et al.
`
`Results and discussion
`
`Modifications with hydrazides
`Carbodiimide -mediated coupling of HA to primary amines failed because the
`acidic pH ( <4.75) required to induce HA -O -acyl urea formation also caused
`protonation of all amine functions. Thus, amines with pKa values (for the
`conjugate acids) in the range of 9 -13 were >99.99% protonated and thus insuffi-
`ciently nucleophilic. The realization that hydrazides possessed sufficiently low
`pKa amines (pKa values of 2 -3 for the conjugate acids), and that efficient reaction
`with EDCI -activated HA could take place in water at pH 4.75, was first
`demonstrated with adipic dihydrazide (ADH). With excess ADH, this reaction
`gave chemically and thermally stable, colourless and spectroscopically well -
`defined materials for both oligosaccharides and high- molecular -mass HA (Figure
`5) [85]. This derivative, called HA -ADH, was the major product, with the cross -
`linked HA- ADH -HA appearing as a minor product. Initial work showed that
`ADH (C6) was superior to both succinic dihydrazide (C4) and suberic
`dihydrazide (C8). Characterization of the HA oligosaccharide adducts by high -
`field solution phase 'H-NMR confirmed that, with ADH, >50% of available
`carboxylates could be modified. The NMR spectra also clearly demonstrated that
`the HA -ADH oligosaccharide adducts retained a reactive hydrazide for further
`derivatization. Indeed, HA -ADH was coupled at pH 8.5 to the N- hydroxysuc-
`cinimide esters of ibuprofen or hydrocortisone hemisuccinate to produce the
`
`Figure S
`
`HA-COOH
`
`+
`
`H2N-NH
`
`NH-NH2
`
`RN=C=NR'
`
`Adipic Dihydrazide
`
`pH -4.75
`RT
`
`urea by- product
`
`0
`HAA'NH -NH
`
`o
`
`o
`
`o
`
`NH-NH2
`
`+
`
`HA
`
`NH-NH
`
`NH-NHHA
`o
`
`11'
`o
`
`HA-ADH
`
`Cross -linked HA- ADH -HA
`
`Chemical derivatization of HA with adipic dihydrazide (ADH) using a
`carbodiimide coupling
`The inset shows the molecular details of a region of HA ADH containing two disaccharide repeat units.
`
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`
`

`

`Chemically modified hyaluronic acid
`
`5 I
`
`Figure 6
`
`HO
`
`NHS, DCC
`Glyme
`
`4°C,16h
`
`cH,
`
`,o
`
`°
`
`N
`
`o
`
`HA-ADH,
`0.1 M NaHCO3
`DMF (1:2)
`
`0
`
`HA
`
`NH-NH
`
`NH`
`
`O
`
`CH3
`
`HA -ADH- Ibuprofen
`
`OH
`
`O
`
`esterase
`
`NH -NH
`
`Ho
`
`1. NHS, DCC,
`OH DMF, rt. 16 h
`2. HA-ADH, DMF,
`0.1 M NaHCO3
`
`00
`
`Hydrocortisone -hemisuccinate
`
`HA -ADH- hydrocortisone
`
`Synthesis of two anti -inflammatory drugs attached to HA -ADH
`Top, HA- ADH -ibuprofen; bottom, HA -ADH- hydrocortisone hemisuccinate. The open arrow indicates the
`
`preferred site of esterase cleavage that would release free hydrocortisone.
`
`corresponding HA- tethered drugs (Figure 6). Finally, exploratory work with four
`commercially available homobifunctional cross -linkers led to the production of
`soluble, cross -linked HA -ADH derivatives, as evidenced by broadened signals for
`the ADH and cross -linker methylenes in the solution NMR [85].
`The solution phase work was then extended to HA with a molecular
`mass of 1.5 MDa. Solid -state 13C -NMR using CP -MAS of lyophilized native HA
`and HA -ADH revealed that both retained largely solution -like structures in the
`solid -state [67], although subtle changes in acetamido orientation and glycosidic
`dihedral angles have been previously discerned [86]. With HA -ADH possessing
`approx. 20 -25% modification of the glucuronates, four homobifunctional cross -
`linkers were used at a molar ratio of 1:1.4 (HA:cross- linker) to create swellable
`hydrogels (Figure 7). The CP -MAS spectra gave maximum sensitivity for the
`determination of the chemical shifts of the carbon atoms of the covalently
`attached bishydrazide tether and cross -linker. Moreover, subtraction of a native
`HA spectrum from the derivatized HA spectrum allowed visualization of a
`residual intensity corresponding to linker groups; calculated chemical shifts
`confirmed the nature of the linkage. Earlier work had indeed shown broadened
`resonances for cross -linkers, indicating disorder. In contrast, the HA resonances
`did not broaden, suggesting a highly ordered, rigid structure. Nonetheless, upfield
`chemical shifts were observed in one of the HA resonances, suggesting an increase
`in stcric crowding resulting from a change in sugar pucker.
`Scanning electron microscopy of lyophilized hydrogels revealed
`macroporous sponge -like materials with varying surface morphologies, but all
`
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`
`

`

`52
`
`G.D. Prestwich et al.
`
`Figure 7
`
`NH2+CI-
`
`CH30
`
`OCH3Ñ
`
`DMS
`
`Na03S,
`
`O
`
`Na03s,
`
`O
`
`O
`
`O
`
`Na03
`
`O
`
`O-N
`0 O
`
`O
`
`o
`
`o
`
`_
`
`SO3Na
`
`BS3
`
`SO3Na
`
`DTSSP
`
`Linkers
`
`O
`
`OJ v '(O0)
`
``
`
`rO'N
`o
`
`sulfo-EGS
`
`SO3Na
`
`HA
`
`O
`
`ÑH"NHÓNHNH2
`
`0.1 M NaHCO3
`
`O
`
`O
`HA NH-NH
`\lf v v NH-NH
`O
`
`NH2.
`
`O
`1'
`
`HA^NH-NH1
`
`NH-NH
`
`O
`
`NH-NH)LHA
`
`o
`
`NH-NH
`
`HA
`
`o
`
`O
`
`NH-NH
`NH2+
`
`,,
`NH-NH
`
`IO
`
`O
`
`O
`
`O
`
`H
`
`A
`
`HA NH-NH
`
`O
`
`O
`
`O
`
`NH-NH - S-S/
`
`Gels
`
`0
`
`NH-NH
`
`HA
`
`p
`
`o
`
`NH-NH 0 O
`p NH-NH
`
`NH-NH
`
`HA
`
`Strategy for cross -linking of HA -ADH to give hydrogels with four different
`homobifunctional cross -linkers
`The open arrow indicates a site for reductive cleavage of the disulphide gel. Similarly, the dark arrows
`indicate hydrolysis of the diester backbone of the HA hydrogel to release HA -bound gel fragments.
`
`showing average pore diameters of 20 to 50 p.m [67]. For example, the HA -ADH-
`DTSSP- ADH -HA (Figure 7) hydrogel had continuous hexagonal -type pores that
`were not interconnected, while HA- ADH -BS3- ADH -HA showed elongated
`
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`

`

`Chemically modified hyaluronic acid
`
`53
`
`pores in a sheet-like network. In preliminary tests for biocompatibility, HA-
`ADH and its cross -linked derivatives have been examined using a proprietary in
`vitro assay of inflammatory potential (Collaborative Laboratories Inc.). Test
`substances are incubated with human neutrophils on a model radiolabelled ECM,
`and neutrophil activation (the first step in inflammatory response) is detected by
`measuring radiolabelled ECM degradation products resulting from enzymes
`released by activated neutrophils. In this system, HA -ADH was found to
`decrease the level of neutrophil activation, i.e. HA -ADH was intrinsically anti -
`inflammatory (D.C. Watkins, J.A. Hayward and B. Costello, personal communi-
`cation).
`
`A. Monohydrazides
`
`Figure 8
`
`H2NHN
`
`o
`
`cr
`N(CH3)3
`
`H2NHN
`
`H2NHN
`
`0
`H2NHN v
`
`'
`
`B. Bishydrazides
`
`o ^ ^ O
`H,NHN" v `s-5 v NHNH2
`
`o
`
`H2NHN
`
`OH
`
`NHNH2
`
`Hs OH O
`
`O
`
`H2NHN O O
`
`n
`
`n
`
`NHNH2
`
`O
`
`NHNH_
`
`H2NHN
`
`C. Polyhydrazides
`
`)L/`N
`
`H2NHN
`
`NHNH2
`
`H2NHN
`
`O
`
`NHNH2
`
`m=1,2,3,6
`
`N O
`
`' NHNH2
`
`H2NHN
`
`H2NHN
`
`NHNH2
`
`ONHNH2
`
`H2NHN O
`
`NHNH2
`
`H2NHN
`
`O
`
`o
`
`NHNH2
`
`0
`
`H2NHN
`
`H2NHN.O
`
`O
`
`NHNH2
`
`Structures of selected hydrazide reagents used for modification of HA
`A monofunctional hydrazides; B. bisfunctional hydrazides; C, polyfunctionol hydrazide cross -linkers.
`
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`

`

`54
`
`G.D. Prestwich et al.
`
`Figure 9
`
`dihydrazide,
`carbodiimide
`
`Native HA
`
`Hydrazido-HA
`
`activated ester
`drug molecule
`
`b bisdìhydrazide cross -link
`--pendant hydrazide functionality
`
`homobifunctional cross -linker
`
`Drug -HA
`
`© drug molecule
`inaccessible functionalities
`
`Cross -linked HA Hydrogel
`
`Homobifunctional cross -links
`
`Cross -linked Drug -HA
`
`drug molecule
`- homobifunctional cross -links
`
`Schematic representation of the stepwise introduction of functionality and
`cross -links to native HA
`A few cross -links (open arrows) occur spontaneously during the original functionalization, while the
`
`majority o f ADH groups (wavy lines) are available for either attachment of reporter or biologically active
`
`groups (spheres) or cross - linkers (filled bars).
`
`We have now extended the scope of the hydrazide technology to include
`branched and heteroatom- containing bishydrazides, as well as a number of
`monohydrazides. The structures of these reagents are illustrated in Figure 8.
`Monohydrazides (Figure 8A) allow the replacement of a defined proportion of
`
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`
`

`

`Chemically modified hyaluronic acid
`
`55
`
`Figure 10
`
`EDCI, pH 4.75
`
`Drug- modified
`polyhydramde
`
`Native HA
`
`Drug - Polyhydrazide
`Cross -linked HA Hydrogel
`
`The polyfunctional cross -linker strategy allows simultaneous introduction
`of cross -linking groups and a pendant chemical functionality for further
`modification
`Alternatively, the bioeffector molecule may be attached to a polyvalent linker after the cross -linking
`
`reaction. Fewer pendant groups can be incorporated using this strategy.
`
`the glucuronate carboxylic acids with sulphydryl, aliphatic, aromatic,
`fluorophoric, cationic or bioactive residues. The resulting products have, respec-
`tively, modified chemical attachment properties, additional hydrophobic patches,
`novel reporter functions, inverted charge properties or bioreleasable payloads
`[87]. The bishydrazides (Figure 8B) were selected or synthesized to enable direct
`cross -linking of HA in a single step using tethers with distinctive chemistries.
`the overall
`Figure
`biologically active ligand and cross -linking to form a bioerodable hydrogel that
`offers slow release of the active agent.
`Polyhydrazides (Figure 8C) have also been synthesized based on
`modifications of dendrimer chemistry using 3-4 mol- equiv. methyl acrylate per
`mole to mono -, di- and triamines [88], followed by hydrazinolysis (J.F. Marecek,
`D.M. Marecak and G.D. Prestwich, unpublished work) [87]. These polyfunc-
`tional cross -linkers allow cross -linking of HA while retaining a pendant
`hydrazide functionality for subsequent (or prior) attachment of a bioeffector
`group possessing an active ester functionality. This modified strategy is illustrated
`in Figure 10.
`
`Optimization of hydrogel synthesis
`The creation of stable hydrogels from HA is challenging. Conditions can be
`selected that result in hydrogels with different physical properties and rates of
`gelation. The important parameters include pH, concentration and nature of metal
`ions present, ratios of HA to EDCI to linker, chemical nature of linker, nature and
`concentration of buffer, and molecular size range and concentration of HA. Table 1
`presents an overview of the reaction parameters involved and their effects on the
`properties of the resulting HA hydrogels (K.P. Vercruysse, D.M. Marecak, J.F.
`Marecek and G.D. Prestwich, unpublished work). Softer, more pourable gels are
`favoured over solid gels by a number of selections: (a) lower concentrations of
`HA (2.5 versus 8.0 mg /ml), (b) a higher pH (approx. 4.75 versus 3.5), (c) a lower
`concentration of Bis -Tris buffer (100 versus 500 mM), (d) fewer equiv. of cross-
`
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`

`56
`
`G.D. Prestwich et al.
`
`Table 1
`
`Soft -pourable gel
`Reagent
`Solid gel
`HA
`2.5 mg /ml
`8.0 mg /ml
`pH =4.7
`0.1 M HCI
`pH =3.5
`100 mM
`500 mM
`Bis -Tris HCI
`Cross -linker
`0.1 mol -equiv.
`1.5 mol -equiv.
`0.5 mol -equiv.
`EDCI
`1 mol -equiv.
`Buffer
`Tris (pKa= 8.1)
`Bis -Tris (pKa= 6.5)
`Reaction parameters and their effects on the physicochemical properties
`of HA hydrogels
`
`linker (0.1 per glucuronate versus 1.5 mol- equiv.), (e) reduced amounts of EDCI
`(0.5 versus 1 mol- equiv.) and (f) the type of buffer (Tris versus Bis- Tris). The
`production of a porous three -dimensional network that is biocompatible,
`bioerodable, localizable, injectable and capable of furnishing viscosupplementary
`physical effects would be the ideal target material.
`In addition to the parameters in the table, higher temperatures
`(50 -75 °C) are often needed to dissolve higher, more hydrophobic bishydrazides
`in water. However, while coupling may improve at higher temperatures, cross-
`linking may be reduced. Co- solvents such as acetone, DMSO, DMF, dioxane and
`alcohols have been used in amounts that do not affect the integrity of the HA
`molecular size range or cause precipitation. While it is preferable to avoid non -
`aqueous solvents in potential medical products, low percentages may assist in
`obtaining suitable hydrogels. The mode and speed of mixing can influence
`gelation. It is difficult to obtain a homogeneous, fast -setting gel with simple
`magnetic stirring. Varying the ratios of HA : bishydrazide: carbodiimide, as shown
`in the table, is one of the most effective routes to altering gel properties.
`Conditions may use from 0.01 to 20 equiv. of bishydrazide per HA glucuronate
`moiety, and from 0.1 to 2 equiv. of carbodiimide per equiv of bishydrazide.
`Finally, the effects of metal ions were examined on the gelation of HA
`solutions (3.0 and 4.5 mg /ml) with 0.1 mol- equiv. of the hexamethylenediamine
`tetrahydrazide cross -linker (Figure 8) and 0.4 equiv. of EDCI (D.M. Marecak,
`unpublished work). Gel formation was attempted in the presence of 200 mM
`MnC12, CaCl2, BeCl2, CoCl2, FeCI or CuCI,. At 4.5 mg /m1 HA, gelation was
`rapid in the presence of Mn2 +, Ca2+ or Bee+ and in the absence of any salts. In the
`presence of Col* the gelation proceeded much more slowly, and in the presence of
`Fee+ or Cult no gels were obtained. With 3.0 mg /ml HA, no gelation occurred in
`the absence of salts; however, in the presence of Mn2+ or Ca2 +, gelations were again
`observed. The firmness of the gels obtained at 4.5 mg /ml decreased in the order
`Ca2+ > Mn2+ > Be2+ > no salt.
`
`Hydrogel degradation by HAse
`In the body, HA is degraded by HAse, which is present in virtually every cell and
`in serum [89,90]. The kinetics