Hyaluronan: Pharmaceutical Characterization and Drug Delivery

Abstract

Hyaluronic acid (HA), is a polyanionic polysaccharide that consists of N-acetyl-D-glucosamine and β-glucoronic acid. It is most frequently referred to as hyaluronan because it exists in vivo as a polyanion and not in the protonated acid form. HA is distributed widely in vertebrates and presents as a component of the cell coat of many strains of bacteria. Initially the main functions of HA were believed to be mechanical as it has a protective, structure stabilizing and shock-absorbing role in the body. However, more recently the role of HA in the mediation of physiological functions via interaction with binding proteins and cell surface receptors including morphogenesis, regeneration, wound healing, and tumor invasion, as well as in the dynamic regulation of such interactions on cell signaling and behavior has been documented. The unique viscoelastic nature of hyaluronan along with its biocompatibility and nonimmunogenicity has led to its use in a number of cosmetic, medical, and pharmaceutical applications. More recently, HA has been investigated as a drug delivery agent for ophthalmic, nasal, pulmonary, parenteral, and dermal routes. The purpose of our review is to describe the physical, chemical, and biological properties of native HA together with how it can be produced and assayed along with a detailed analysis of its medical and pharmaceutical applications.

Keywords :

• Analysis
• Drug Delivery
• Hyaluronan
• Physiochemical Properties
• Topical

Hyaluronic acid (HA), discovered in bovine vitreous humor by Meyer and Palmer in 1934, is most frequently referred to as hyaluronan due to the fact that it exists in vivo as a polyanion and not in the protonated acid form. HA is ubiquitous in that it is distributed widely in vertebrates and present as component of the cell coat of many strains of bacteria (e.g., A. Streptococcus) (Laurent 1970). Commercially produced HA is isolated either from animal sources within the synovial fluid, umbilical cord, skin, and rooster comb, or from bacteria through fermentation or direct isolation. The molecular weight of HA is heavily dependent on its source; however, refinement of these isolation processes has resulted in the commercial availability of numerous molecular weight grades extending up to a maximum of 5,000,000 daltons (Milas et al. 2001). Extensive studies on the chemical and physicochemical properties of HA and its physiological role in humans, together with its versatile properties, such as its biocompatibility, nonimmunogenicity, biodegradability, and viscoelasticity, have proved that it is an ideal biomaterial for cosmetic medical and pharmaceutical applications.

Several of HA's important physicochemical properties include molecular weight dependence. Therefore discrete differences in function over the wide range of commercially available molecular weights enables HA to used in a diverse set of applications. In addition, chemical modification of hyaluronan can confer more extensive applications. While retaining biocompatibility and biodegradability, chemical modification can produce a more mechanically and chemically robust material. Methods detailing the synthetic alteration of HA and the resulting applications in cosmetic, medical, and pharmaceutical applications have been reviewed previously (Larsen and Balazs 1991; Denlinger 1998; Band 1998; Lapcik et al. 1998; Vercruysse and Prestwich 1998; Adams et al. 2000; Moreland 2003; Campoccia et al. 1998) and are not included in this article. The purpose of our review is to describe the physical, chemical, and biological properties of native HA together with details of its medical and pharmaceutical uses.

PHYSICAL AND CHEMICAL PROPERTIES OF HA

Chemical Structure and Stability

The precise chemical structure of HA, which comprises repeating units of D-glucoronic acid and N-acetyl-D-glucosamine, was first determined by Weissman and Meyer (1954). Further studies (Laurent 1970; Laurent and Fraser 1992) have shown the primary structures of the polysaccharide to be an unbranched linear chain with the monosaccharides linked together through alternating β_1,3 and β_1,4 glycosidic bonds (Scott and Heatley 1999).

Data obtained from viscosity measurements, light scattering, and ultracentrifugation suggest that HA molecules assume an expanded random coil structure in solution (Lapcik et al. 1998). However, these techniques only provide a measure of the bulk properties and thus are indicative of the mean contribution of a large number of molecules within a polydispersive distribution and do not necessarily provide molecular information. However, nuclear magnetic resonance spectroscopy (NMR), X-ray diffraction, and molecular modeling all provide evidence of the existence of ordered structure in HA molecules (Lapcik et al. 1998; Scott 1984; Scott and Heatley 1999; Day and Sheehan 2001). The data obtained from these techniques suggest that up to 5 hydrogen bonds exist between two neighboring disaccharide units and the secondary structures are formed as tape-like 2-fold helices by twisting each disaccharide unit through 180°. In addition, secondary hydrophobic faces exist within the structure, formed by the axial hydrogen atoms of about 8 CH groups on the alternating sides of the secondary structure. Such hydrophobic patches, in turn, energetically favor the formation of meshwork-like β -sheet tertiary structure as a result of molecular aggregation (Figure 1B). The tertiary structure is stabilized further by the presence of intermolecular hydrogen bonding (Scott and Heatley 1999). The hydrophobic and hydrogen bonding interactions, countering electrostatic repulsion, enable large numbers of molecules to aggregate leading to the formation of molecular networks (matrices) of HA (Figure 1C).

FIG. 1 The primary, secondary, and tertiary structures of HA in solutions. (A) The primary structure of HA consists of repeating dissacharide units of D-glucoronic acid and N-acetyl-D-glucosamine with up to 5 hydrogen bonds existing between each two neighboring disaccharides. The secondary structures are formed as tape-like 2-fold helices by twisting each disacchride unit through 180° compared with those ahead and behind it in the chain. (B) The β -sheet tertiary structure tertiary structure is energetically stabilized by interactions between hydrophobic patches hatched and intermolecular hydrogen bonding between the acetamido ▪and □ and carboxylate groups (• and ○). (C) Schematic networks of HA molecules as a results of intermolecular aggregation (Reprinted with permission from Scott and Heatley 1999).

Such an ordered and rigid structure is more stable than any random state that may exist in solution although it does possess varying degrees of flexibility and may undergo interconversion between different conformation states. For example, Scott and Heatley (2002) reported that the stability of the tertiary structure of HA appeared to be marginal in physiological solutions. In this study, the mesh-like HA molecules were susceptible to reversible de-aggregation, with changes to an unordered structure (denaturation) occurring in response to cooling, warming, or pH. Such denaturation was irreversible upon enzymatic degradation and chemical modification. The delicate equilibrium between ordered and unordered structure is assumed to be a consequence of steric, hydrophobic, and particularly hydrogen bonding interactions, which occur between HA chains and water, although detailed dynamics in molecular terms have yet to be elucidated.

Apart from the inherent instability of highly ordered secondary and tiertary structures, the primary structure of HA also is susceptible to degradation under various conditions, including mechanical, ultrasonic, pH, thermal, free radical, and enzymatic stresses (Lapcik et al. 1998). The dependence of HA's stability on pH has been reported by Tokita and Okamoto (1995) who showed that HA possesses the highest stability at neutral pH, whereas they found it to be more labile in acidic solution than basic and to be less stable at higher temperatures. In general, the degradation of a HA solution under pH, thermal, and free radical stresses has been reported to occur randomly (Reed and Reed 1989; Tokita and Okamoto 1995; Vercruysse et al. 1995; Lowry and Beavers 1994). In contrast, the depolymerization that occurs following ultrasonic treatment has been reported to occur in a nonrandom manner (Hawkins and Davies 1996) and to be mechanical in origin (Basedow and Ebert 1977; Miyazaki et al. 2001).

Mechanical depolymerization of HA also has been demonstrated to occur using a rotational viscometer, where the molecular weight of HA in a diluted solution was found to decrease with increasing shear stress (Miyazaki et al. 1998). In this study, the authors found that the position of the cleavage of the HA chain depended upon the shear rate loaded and ionic strength. The same authors utilized ultrasonication in specific ionic conditions to depolymerize HA to form controlled molecular weights (Miyazaki et al. 2001). The degradation of HA solution induced by stresses is of pharmaceutical relevance. For example, upon freeze-drying, HA has been reported to be susceptible to depolymerization (Pigman et al. 1961; Wedlock et al. 1983; Doherty et al. 1994; Tokita et al. 1997). During the freeze-drying process, the stability of HA depends upon the form of HA employed, with sodium HA being more stable than sodium-free HA. The instability of HA has been attributed to the production of free radicals during this process and can be minimized by the presence of free radical scavengers (Wedlock et al. 1983; Tokita et al. 1997).

Physicochemical Properties

HA and its associated networks have many physiological roles that include tissue and matrix water regulation, structural and space-filling properties, lubrication, and a number of macromolecular functions (Balazs 1998). Changes in the ordered structures of HA can lead to major alterations in its physicochemical (e.g., viscoelastic and mechanical) properties since they are largely dominated by the nature of the secondary and tertiary structures. In the solid state when prepared at a low pH, HA has been found by X-ray diffraction to possess double helical structures (Arnott et al. 1983) that are markedly different from the structures of HA formed in aqueous solution. The effect of water on the structure and dynamics of HA is a consequence of the strong hydrogen-bonding that exists between the two molecular species. These interactions are illustrated by the highly inhibitory effect on the freezing of water conferred by 0.5 and 3.0% w/w HA in aqueous solution, as determined by modulated differential scanning calorimetry. Results have shown that 1 g of HA could lead to the presence of about 3.65 g of nonfreezing water during the freezing process (Liu and Cowman 2000).

The number of hydrogen bonds between water molecules and the hydrogen bond accepting atoms of HA has been calculated using molecular dynamic simulations to be between 10 and 15 per disaccharide unit of HA (Kaufmann et al. 1998). As such, when compared with such other polysaccharides as alginate, carrageenan, guar gum, locust bean gum, and tragacanth gum, HA has a much higher capacity to bind water (Budiaman and Fennema 1987; Cowman et al. 1998).

HA in aqueous solution has been reported to undergo a transition from Newtonian to non-Newtonian characteristics with increasing molecular weight, concentration, or shear rate (Gribbon et al. 2000). In addition, the higher the molecular weight and concentration of HA, the higher the viscoelasticity the solutions possess (Gibbs et al. 1968; Gribbon et al. 2000; Karim et al. 1996; Kobayashi et al. 1994; Fujii et al. 1996). The networks of gelling HA are initially formed by weak interactions between HA chains and these can be readily disrupted upon increasing shear rate leading to shear-thinning (Gribbon et al. 2000). The viscoelasticity of HA in aqueous solution also is affected by the inclusion of a small molecular agent (Kobayashi et al. 1994; PasqualiRonchetti et al. 1997; Mo et al. 1999; Ghosh et al. 1994). For example, the presence of phospholipids, guanidine, and sodium chloride leads to decreases in both elastic and viscous moduli while the addition of sugars appears to promote structure.

The effect of these compounds on the viscoelasticity is due to the disruption of intermolecular interactions and reduction in the entanglement between HA molecules. Guanidine and sodium chloride act by shielding electrostatic repulsion between HA molecules (Mo et al. 1999) whereas phospholipids provide competition for the hydrophobic binding between HA molecules (Ghosh et al. 1994). When sugars such as sucrose or glucose were added to HA solutions, both the radius of gyration and hydrodynamic radius of HA were reduced such that the interactions between HA molecules were enhanced; this resulted in an increase in the storage and loss of moduli (Kobayashi et al. 1994; Mo et al. 1999).

The viscoelastic properties of HA in aqueous solution are pH-dependent. HA has a pKa value of about 3.0 and therefore a change in pH will affect the extent of ionization of the HA chains. A shift in ionization affects the intermolecular interactions between the HA molecules which changes the rheological properties of the compound. Gibbs et al. (1968) found that HA in solution possesses entirely different viscoelastic properties at pH 2.5 compared with those at either pH 1.5 or 7.0 with the elasticity be higher at pH 2.5 than at either 1.5 or 7.0 (Gibbs et al. 1968). In physiological solution, the carboxylic groups within D-glucuronic acid of HA are completely ionized with the charges being sited about 1 nm from each other. However, the charges on HA chains are sensitive to specific ionic conditions, which may cause alterations in the bulk solvents, and the inter and intramolecular interactions. For example, with the addition of different ions (Ca^{2 +}, Mn^{2 +}, Na⁺, K⁺, and Mg^{2 +}), the visocoelasticity of HA solutions was different because of the different effects conferred by the ions on the HA chain flexibility (Sheehan et al. 1983; Gribbon et al. 1999; Gribbon et al. 2000).

HA in combination with other glycosaminoglycans (GAGs) such as dermatan sulphate (Koshiishi et al. 1999), chondroitin sulphate, and keratin sulphate (Poggi et al. 1999) are prominent in tissues such as the skin. HA is found to exist together with protein cores (aggrecans) to which the other members of the GAG family are attached. As its name implies, aggrecan is composed of very large proteoglycan aggregates. The most important property of these molecules is their ability to bind to water and this induces the proteoglycans to become hydrated to such an extent that a gel-like system is formed. The networks of proteoglycan-HA aggregates shift the Newtonian region to lower shear rates and these gels have an increased dynamic viscoelasticity relative to HA-HA networks.

Apart from the unique rheological properties, HA solutions exhibit mucoadhesive properties (Saettone et al. 1989; Durrani et al. 1995; Pritchard et al. 1996; Lim et al. 2000). Saettone et al. (1989) and Durrani et al. (1995) found that the degree of mucoadhesion exhibited by HA was both pH- and molecular-weight dependent. Increasing the molecular weight of HA from 134,000 to 4 million Da or decreasing pH from neutral to acidic was found to promote adhesion.

Biological Properties

HA is found in almost all vertebrate organs, but most abundantly in the extracellular matrix of soft connective tissues. HA is particularly abundant in mammalian skin where it constitutes a high fraction of the extracellular matrix of the dermis (Juhlin 1997; Tammi et al. 1994). The estimated total amount of HA in human skin has been reported to be 5 g (Banks et al. 1976), about one-third of the total amount of HA believed to be present within the entire human body (Laurent 1995). HA is present in both the dermis (∼ 0.5 mg/g wet tissue) and the epidermis (∼ 0.1 mg/g wet tissue). Interestingly, while the dermis consists primarily of extracellular matrix with a sparse population of cells, the epidermis is the reverse; the keratinocytes fill all but a small percentage of the tissue. The actual concentration of HA in the matrix around the cells in the epidermis (estimated to be 2-4 mg/ml) is an order of magnitude higher than in the dermis (estimated to be ∼ 0.5 mg/ ml). Thus, the matrix around keratinocytes may have a HA concentration as high as that in umbilical cord (∼ 4 mg/ml). In addition, Sakai et al. (2000) have shown that HA is derived from keratinocytes beneath the stratum corneum and is present in the normal stratum corneum (Sakai et al. 2000).

HA biosynthesis occurs at the plasma membrane under control of HA synthase (Has) that has 3 isoforms: Has1, Has2, and Has3 (Itano and Kimata 2002). In addition, apart from the polymerization of HA chains on the inner face of the cell membrane, the proteins also are responsible for the extrusion or translocation of the polymer through the membrane to the extracellular face of the cell. Although any of the Has proteins are sufficient for HA production, the three isoforms possess different enzymatic properties including kinetic characteristics and the size of the HA produced. Has1 and Has2 proteins are responsible for the synthesis of high molecular weight HA (∼ 2 × 10⁶ Da) with the latter more active catalytically than the former, whereas Has3 protein possesses the highest activity but polymerizes low molecular weight HA (< 3 × 10⁵ Da) (Spicer and Nguyen 1999). The different molecular weight of HA chains can lead to different effects on cell behavior.

The turnover of HA has been found to be as important as its synthesis since the balanced metabolism of HA contributes to its physiological functions (Lee and Spicer 2000). In humans, the half-life of HA in tissues ranges from about 1 day in the epidermis (Tammi et al. 1991) up to 70 days in the vitreous body of the eye (Fraser et al. 1997). HA turnover within a tissue occurs either by local degradation or release from the tissue into the lymphatic/vascular systems. Generally, the removal of HA from the tissues takes place within the lymph nodes after entering the lymphatic system, where about 10% of the HA is transported to the circulation and cleared in the liver or the kidney (Fraser et al. 1998). The degradation of HA within the lymph nodes uses receptor-mediated endocytosis and at least two receptors, hyaluronan receptor for endocytosis (HARE) (Zhou et al. 2000) and LYVE (lymphatic vessel endothelial HA receptor [Banerji et al. 1999; Prevo et al. 2001]), have been identified as being involved.

The main clearance of circulating HA, either intravenously injected or transported from the lymphatic system, takes place rapidly in the liver also via receptor-mediated endocytosis resulting in a HA half-life of less than 10 min in humans (Lebel 1991). In contrast, the degradation of HA in the kidney is achieved via cellular uptake and lysosomal degradation by specific enzymes in an acid cellular compartment (Wuthrich 1999; Knudson et al. 2002). Local turnover of matrix HA within the tissues generally occurs again through receptor-mediated endocytosis via the action of a specific binding protein, CD44 (Menzel and Farr 1998). However, some may depolymerize to short HA chains through the action of hyaluronidase or chemical free radicals (Ng et al. 1992). Once internalized via CD44 binding, HA is degraded mainly within lysosomes to small fragments.

Many physiological functions of HA are thought to relate to its molecular characteristics, including its physiochemical properties, its specific interactions with hyaladhereins, such as CD44 and RHAMM (receptor for HA-mediated motility), and its mediating effect on cell signaling and behavior (Tool 2001). HA in physiological solution can act as an osmotic buffer, space filler, lubricant, and molecular sieve and play a role in maintaining the water content (Fraser et al. 1997). Small molecules such as water, electrolytes, and nutrients can freely diffuse through the pores formed within a solution/gel network while macromolecules such as large proteins and pathogens are partially excluded from the domain. However, networks of HA chains are highly dynamic in nature and constantly move in solution, leading to continuous changes in pore size. Thus, in certain situations even large macromolecules such as proteins have the potential to pass through a HA network. Water homeostasis is attributable to the strong water-HA interaction, which may be responsible for the regulation of moisture content in the epidermis/stratum corneum (Sakai et al. 2000).

Recently, an understanding of the role of HA in the mediation of physiological functions via interaction with binding proteins and cell surface receptors has emerged. The involvement of HA in many biological processes, including morphogenesis, regeneration, the regulation of cell signaling and behavior, wound healing, and tumor invasion have been described in numerous recent reviews (Cichy and Pure 2003; Ponta et al. 2003; Tammi et al. 2002; Day and Prestwich 2002; Turley et al. 2002; Toole et al. 2002). For example, HA binding to CD44 has been reported to lead not only to the removal of the polymer from the tissues, but also the initiation of the signaling cascade that contributes to cell motility and proliferation. However, the exact mechanisms by which different signals are activated have not yet been defined completely (Goodison et al. 1999; Tammi et al. 2002). In addition, the interactions of HA with RHAMM have been reported to mediate cell migration and proliferation (in both normal and tumor cells) along with being involved in the modulation of acute and chronic inflammation processes in man (Turley et al. 2002).

ANALYSIS OF HA

Quantification

The lack of an ultraviolet (UV) or fluorescent chromophore (Toida et al. 1997) within HA's native structure, its large molecular size, and wide molecular weight distribution make accurate and sensitive quantification of this molecule problematic. With the exception of Grimshaw et al. (1994) who directly detected HA from the eye vitrious humor using low wavelength UV detection, the majority of reported chromatographic and colorimetric methods conjugate, react, or lyse HA to facilitate its analysis.

Conjugation

The European Pharmacopoeia (2002) describes a simple colorimetric assay in which the glucuronic acid content of the polysaccharide is determined using a reaction with carbazole. This method was originally adapted from the work of Bitter and Muir (1962). The carbazole modified HA absorbs UV light at 530 nm and produces a linear calibration in the range 6.5–65 μ g g^{− 1}. A molar ratio calculation is subsequently used to determine the HA concentration.

Although colorimetric analysis is very simple and quick to perform, it can be affected by impurities and therefore chromatographic methods are more suitable for the analysis of complex mixtures containing HA (Asteriou et al. 2001). Platzer et al. (1999) highlighted this problem when they compared capillary electrophoresis (CE) and the carbazole reaction to quantify HA within simple gel formulations. CE was capable of resolving the intact polysaccaride from the formulation excipients and produced a linear calibration curve (r² > 0.999) over a 0.01–5 mg ml^{− 1} concentration range. Compared with the colorimetric assay, CE recovered significantly more of the polysaccaride from the gel formulations and furthermore was capable of quantifying the preservative in the formulations, p-hydroxybenzoic acid.

Gel filtration commonly is used to quantify HA in its native form. Chaidedgumjorn et al. (2002) used suppressed conductivity detection to detect both HA polysaccarides and oligomers down to 6.78 ng (100 K M_w) on two coupled TSKgel G3000SWXL columns. However, this method was not capable of fully resolving a mixture of intact HA polysaccharides over a 70-min run time. Toida et al. (1997) proposed combining HA with copper (II) to enhance its analysis. This group coupled GFC (HW40F, Toyo-Pear) with UV detection as the copper provided HA with optical properties. A highly resolved HA-copper(II) peak was eluted within 12 min, however, the effects of HA's molecular weight, polydispersity, or interfering analytes on the copper(II)-HA complex were never investigated in this study.

Digestion

Digesting HA into its constitutional disaccharide units prior to quantification offers analytical advantages in terms of detection sensitivity. Volpi (2000) used chondroitin sulphate lyases to convert HA into unsaturated disaccharides that can be resolved and quantified using high performance liquid chromatography (HPLC). This method used flourimetric derivatization with dansylhydrazine originally described by Shinomiya et al (1989). Volpi (2000) completely separated the disaccarides as a result of the HA digestion and detected down to 50 picomol. Karamanos and Hjerpe (1997) have since reported detection limits for unsaturated disaccharides using similar CE methodology within the attomole range.

Molecular Weight and Molecular Weight Distribution

HA is a homogenous polysaccharide generally with a very high molecular mass. However, in vivo its continual turnover can lead to the existence of oligosaccarides and even smaller molecular weight fragments. Therefore, HA most commonly exists as a population of polysaccharide chains with a wide range of molecular weights (10⁵–10⁷) (Kakehi 2003). Both the average molecular weight and the molecular weight distribution of HA can influence many of its physicochemical properties, e.g., its viscoelastic nature (discussed previously). This has lead to the classification of the commercially produced material into more defined molecular weight fractions compared with the wide distributions found in vivo, thus allowing grading the materials purity, physical, and chemical characteristics.

The importance of molecular weight to HA has led to several techniques developed to measure both the average molecular weight and distribution of HA. These include sedimentation, ultracentrifugation (Laurent and Gergely 1955; Ogston and Stainer 1950, 1951, 1952), light scattering (Reed 1989; Blumberg 1954; Laurent 1960), diffusion (Laurent 1960), osmometry, gel electrophoresis, viscometry, and chromatography. However, only the most commonly employed techniques, gel electrophoresis, chromatography, and viscometry are discussed in depth in this review.

Gel Eletrophoresis

Gel eletrophoresis can be used to determine both the molecular weight and molecular weight distribution of HA, although it is only possible to attain qualitative or semiquanitative data using this method. The earliest methods of gel electrophoresis used cellulose acetate membranes and Alcain blue staining (Seno et al. 1970). However, high porosity agarose gels are now more commonly used as illustrated by Lee and Cowman (1994) This group separated HA from human knee joint synovial fluid using a nonspecific cationic dye Stains-All (3, 3′ -dimethyl-9-methyl-4, 5, 4′, 5′ -dibenzothiacarbocyanine) as a marker for HA detection. High molecular weight HA standards (characterized by light scattering) were used as molecular weight markers and the samples were run on a 0.5% agarose gel. In this method loading 4 μ g of HA (< 0.5 mg/ml) allowed separation of very high molecular weight HA samples up to 6 × 10⁶ Da. The principles from Lee and Cowmans work have been further developed by several other groups (Armstrong and Bell 2002; Volpi and Maccari 2002). In addition, How and Long (1969) characterized the polydispersity of HA from human synovial fluid using electrophoresis by fractionation of the 0.1% agarose gels. Additional details on the electrophoretic analysis of HA, chemically modified HA, and HA oligomers are discussed in a review by Kakehi (2003).

Chromatography

Estimation of molecular weight of HA samples using gel filtration chromatography (GFC) or size exclusion chromatography requires the use of molecular weight standards such as polyethylene oxides (Beaty 1985), dextrans (Praest 1997), and pullulans (Iqbal 1997) because HA standards having well-defined molecular weights and narrow molecular weight distributions are not commonly available. Yeung and Marecak (1999) question the use of polymer standards such as pullulans or dextrans to generate a relationship between retention volume and log of molecular weight because of the unique solution conformation of HA.

HA's polydisperse nature has precluded its direct molecular mass analysis using matrix-assisted laser desporption ionization mass spectroscopy (MALDI-MS) because the technique underestimates the molecular weight of such molecules (Jackson et al. 1996). However, Adam and Ghosh (2001), have suggested that coupling gel filtration chromatography with MALDI-MS can resolve this problem. These researchers showed that the weight average molecular weight (Mw) and number average molecular weight (Mn) obtained for 6 of the 7 standards using preseparation with GFC were in good agreement with the reported values. Another novel molecular weight assessment assay for HA is anionic exchange chromatography. Karlsson and Bergman (2003) reported the molecular weight distribution of HA samples in the range of 0.1 × 10⁶ to 5 × 10⁶ Da using this technique.

Viscometry

Static off-line bulb tube viscometry does produce reliable dynamic viscosity measurements across a wide range of HA samples (Mendichi et al. 1998). However, bulb viscometry can be cumbersome and unsuitable for quality control. Therefore, the viscosity average molecular weight (M_v) is more frequently determined for HA, which is derived from the intrinsic viscosity [η] using the following Mark-Houwink equation

Both Karim, 1996 and Laurent, 1960 describe HA molecular weight characterization using viscometry. The measurement was carried out over a range of concentrations (for example, 0.15–5.6 × 10^{− 3} g/ml) and intrinsic viscosity was estimated using Huggins and Kraemer plots. Values of K and α have been determined for HA in both 0.2 M and 0.5 M NaCl by Cleland and Wang (1970). Unsurprisingly, values of K and α depended on the ionic strength (Shimada and Matsumura 1975; Cleland and Wang 1970) and the pH (Balazs, Cowman, and Briller 1983) of the HA solutions. Gura et al. (1998) also showed that the coefficients K and α are affected by the magnitude of shear applied to the material. This work postulated that large molecular weight HA samples require a low shear viscometer such as the Zimm-Crothers machine to attain accurate coefficient values, as the onset of shear-dependent viscosity shifts to a lower shear rate due to the re-enforced entanglement of the intermolecular domains in high weight HA (Gura et al. 1998).

MEDICAL APPLICATIONS OF HA

Treatment of Osteoarthritis

Under normal physiological conditions, HA is one of the principal components of synovial fluid present at concentrations between 1.4–4 mg/ml (Sundblad 1965). The viscoelasticity of the HA matrix is responsible for conferring lubrication and mechanical support to joints. However, the molecular weight of HA in osteoarthritic joints can decline to 0.5 million Daltons (from 4–5 million Daltons in the normal joints) as a result of the presence of proinflammatory cytokines, free radicals, and proteinases. In addition, the membrane permeability of the synovial vasculature increases in response to inflammation or injury, leading to increases in the infiltration of plasma fluids and in turn decreases in concentration of HA in the joints. The decrease in the concentration and/or the molecular weight of HA can result in changes in the viscoelasticity of the synovial fluid, leading to joint dysfunction. Therefore, the rationale for injecting purified high molecular weight HA into the joints is to restore the desirable rheological properties, alleviating some of the symptoms of osteoarthritis (Balazs and Denlinger 1993, 1989).

Intra-articular injection of HA is approved for the treatment of osteoarthritis in many countries including the USA, Canada, Japan, and some parts of the European Union. Commercially available products for this application contain HA with different molecular weight distributions. For example, the molecular weights of Hyalgan® (Fidia, Italy), Artz® (Seikagaku, Japan), and Orthovisc® (Anika, USA) are 0.5–0.73, 0.6–1.2, and 1.0–2.9 million Daltons, respectively. A summary of the commercial products that use HA can be found in Table 1.

TABLE 1 Summary of the medical applications of HA

Disease state	Applications	Commercial products	Publications
Osteoarthritis	Lubrication and mechanical support for the joints	Hyalgan®(Fidia, Italy), Artz® (Seikagaku, Japan) Orthovisc® (Anika, USA) Healon®, Opegan®, and Opelead®	Hochburg 2000; Altman 2000; Dougados 2000; Guidolin et al. 2001; Maheu et al. 2002; Barrett et al. 2002; Miltner et al. 2002; Tascioglu 2003; Uthman et al. 2003; Kelly et al. 2003; Hamburger et al. 2003; Kirwan 2001; Ghosh et al. 2002; Mabuchi et al. 1999; Balazs 1998; Fraser et al. 1993; Zhu et al. 2003.
Surgery and wound healing	Implantation of artificial intraocular lens Viscoelastic gel	Bionect®, Connettivina® and Jossalind®	Ghosh 2002; Risberg 1997; Inoue et al. 1993; Miyazaki et al. 1996; Stiebel-Kalish et al. 1998; Tani et al. 2002; Vazquez et al. 2003; Soldati et al. 1999; Ortonne 1996; Cantor et al. 1998, Turino et al. 2003.
Embryo implantation	Culture media for the use of in vitro fertilization	EmbryoGlue® (Vitrolife, USA)	Simon et al. 2003; Gardner et al. 1999; Vanos et al. 1991; Kemmann 1998; Suchanek 1994; Joly et al. 1992; Gardner 2003; Lane 2003; Figueiredo 2002; Miyano 1994 Kano et al. 1998; Abeydeera 2002; Jaakma et al. 1997, Furnus et al. 1998; Jang 2003.

The therapeutic effects of HA injection products on osteoarthritic joints have been extensively investigated in vitro, in vivo, and clinically (e.g., Hochberg 2000; Altman 2000; Dougados 2000; Guidolin et al. 2001; Maheu et al. 2002; Barrett and Siviero 2002; Miltner et al. 2002; Tascioglu and Oner 2003; Uthman et al. 2003; Kelly et al. 2003; Hamburger et al. 2003). Results from these studies indicated that HA confers pain relief and functional improvements. The analgesic effect of viscoelastic HA was found to be comparable or superior to either that obtained after the administration of either corticosteroids (Altman 2000; Kirwan 2001; Guidolin et al. 2001) or nonsteroidal anti-inflammatory drugs (NSAIDs) (Hochberg 2000). However, the mechanism by which HA confers therapeutic effects on osteoarthritic joints appears to be unclear (Ghosh and Guidolin 2002) despite the postulation of Balazs and Denlinger (1993) that it acts as a viscosupplement, the reason it was first utilized in intra-articular injections. In the short term, HA can confer mechanical and lubricating effects, independent of the variation in the molecular weights of marketed HA preparations (Mabuchi et al. 1999; Balazs 2003) such that symptomatic relief can be elicited. The controversy about the role of viscoelasticity in the efficacy of HA for the treatment of osteoarthritis results from the rapid clearance of HA from the joint relative to its long duration of therapeutic effectiveness (Ghosh and Guidolin 2002).

The benefit from HA injections after a few weeks therapy has been reported to last up to 2 months (Altman 2000), which is inconsistent with the rapid local clearance of HA. In vivo studies found that the half-lives of injected HA into the normal joint knees were less than 1 day (Laurent and Fraser 1992), whereas those into the osteoarthritic joints were even shorter (Fraser et al. 1993). These results suggest that HA might confer its clinical benefits at least partly due to pharmacological effects. The pharmacological role of HA may be related to its anti-inflammatory and immunoregulatory properties, which are dependent upon its molecular weight, although detailed mechanisms by which the clinical benefits occur are yet to be established (Ghosh and Guidolin 2002; Zhu and Granick 2003).

Surgery and Wound Healing

HA is used routinely in eye surgery to facilitate the implantation of artificial intraocular lens, and commercially available products include Healon®, Opegan®, and Opelead®. HA also is used in surgical procedures as a viscoelastic gel (for example, Bionect®, Connettivina®, and Jossalind®) to aid healing and the regenerative processes of surgical wounds (reviewed in Balazs and Denlinger 1993; Lapcik et al. 1998; Ghosh and Jassal 2002). The applications of HA in eye surgery mainly is due to its viscoelasticity; its high viscosity at low shear enables the HA solution to be maintained in the operative space, while the shear thinning behavior assists both the protection of ocular cells from damage (acting as a lubricant) and ensures its ease of injection and removal (Balazs and Denlinger 1993; Risberg 1997).

Apart from acting solely as a viscoelastic substance, HA appears to possess a range of biological activity since it promotes corneal (Inoue and Katakami 1993; Miyazaki et al. 1996; Stiebel-Kalish et al. 1998; Tani et al. 2002), diabetic foot (Vazquez et al. 2003), nasal mucosal (Soldati et al. 1999), and venous leg ulcer (Ortonne 1996) wound healing. Miyazaki et al. (1996) suggested that the corneal wound healing effect conferred by HA is partially due to its ability to promote the growth of corneal epithelial cells. In addition, HA also has been reported to decrease neutrophil elastase-induced alveolar injury after inhalation (Cantor et al. 1998). Aerosolization of 0.1% w/v HA solution to Syrian hamsters for 50 min significantly reduced neutrophil elastase-induced alveolar injury of the animals relative to that found in the animals exposed to aerosolized water alone. The authors suggested that the protective mechanism was due to specific binding between HA and lung elastic fibers (Cantor et al. 1998). However, the detailed molecular biological mechanism by which HA facilitates wound healing has yet to be identified (Turino and Cantor 2003).

Embryo Implantation

HA has been reported to be a suitable replacement for albumin in culture media for in vitro fertilization (Simon et al. 2003; Gardner et al. 1999). The utilization of albumin (human serum albumin for human use) in culture media during in vitro fertilization is not only due to its ideal physical properties such as viscosity, but also because it allows easy handling of the embryo, by preventing it adsorbing to the surface of the culture dish (Simon et al. 2003). However, blood-derived albumins may be contaminated with prions and viruses, leading to potential disease transmission. As a result, the utilization of a functional albumin substitute is now preferred (Vanos et al. 1991; Kemmann 1998). HA, as an endogenous component present in the human uterus at a high level (Suchanek et al. 1994), has been found to be compatible with animal and human embryos. The presence of HA as a substitute of albumin has not resulted in any detrimental effects on the development of frozen-thawed mouse and sheep embryos (Joly et al. 1992). In addition, the presence of HA in culture media could act as a cryopreservative to human embryos by maintaining their viability during thawing (Gardner et al. 2003).

More studies have investigated the effect of HA in murine (Gardner et al. 1999; Lane et al. 2003; Figueiredo et al. 2002), porcine (Miyano et al. 1994; Kano et al. 1998; Abeydeera 2002), and bovine (Jaakma et al. 1997; Furnus et al. 1998; Jang et al. 2003) embryo development and viability after culture in HA-based media. Such studies have demonstrated no differences between HA and albumin with regard to the efficiency of in vitro blastocyst production from in vitro-matured oocytes, and they have thus opened the possibility of including HA in culture media as a substitute of albumin. In a recent clinical study, Simon et al. (2003) found that HA-based human embryo transfer medium leads to a comparable pregnancy and implantation rate when compared with albumin based medium (clinical pregnancy and implantation rates of 62.5% and 34% using the former versus 52% and 26.8% using the latter). summarizes both the medical applications and the commonly used commercial preparations containing HA used within this field.

HA IN DRUG DELIVERY

Dermal Drug Delivery

The physiological function of the stratum corneum, the outermost and nonviable layer of the skin, is to act as a protective barrier for the body and as such it is particularly effective at preventing the permeation of drug molecules into and across the skin for dermal (local) and transdermal (systemic) delivery. Although extensive studies are being performed into the latter, relatively few investigations have been conducted into the development of delivery systems for drug targeting to the skin, although HA is one such system that receiving increasing attention.

However, recently a formulation marketed as Solaraze was approved in the United States, Canada, and most of the European countries for the topical treatment of actinic keratoses (AK), the third most common skin complaint in the U.S. (Fieldman et al. 1998; Moy 2000; Del Rosso 2003; Jarvis and Figgitt 2003). AK, relatively common skin lesions that result from excessive sun exposure, are most common on the scalp, face, and/or dorsum of the hands in light-skinned persons aged ≥ 50 years, particularly those with a history of occupational exposure to the sun. AK now are considered to be synonymous with squamous cell carcinoma (SCC) in situ with growing evidence that AK and SCC lie on a clinical, histological, cytological, and molecular continuum (Schartz 1997).

Although the exact mechanism of action of diclofenac in the Solaraze® formulation remains unclear, it has long been established that some NSAIDs have an antitumorigenic effect (Panie 1981; Braun et al. 1989; Falk et al. 1992) that may be attributable to reduced PGE₂ synthesis. PGE₂ is known to attenuate cell-mediated immunity; in particular this prostaglandin inhibits natural killer cell cytotoxicity (Brunda et al. 1980), mitogen-induced lymphocyte proliferation (Tilden and Balch 1982), and macrophage proliferation and antitumor cytotoxicity (Elliott et al. 1988).

The beneficial effect of HA on the dermal delivery of diclofenac also has been rigorously investigated in vitro, in vivo, and in clinical studies (Brown et al. 2002, 1995a, 1995b, 1995c; Brown and Martin 2001; McEwan and Smith 1997; Wolf et al. 2001; Nazir et al. 2001; Brown and Moore 1996; Lin and Maibach 1996). Brown et al. 2002, 1995c and Brown and Martin (2001) have shown in vitro that HA enhances significantly both the partitioning of diclofenac into human skin and its retention and localization in the epidermis when compared with an aqueous control, other glycoseaminoglycans, and other commonly used pharmaceutically acceptable gelling agents (Figure 2). Such results have been confirmed in other studies in which HA was shown to minimize the percutaneous absorption of diclofenac (McEwan and Smith 1997; Wolf et al. 2001; Brown et al. 1995b) indicating the formation of a depot or reservoir of drug in the epidermis (Brown et al. 1995a; Lin and Maibach 1996). These data have been supported by preclinical studies where radiolabelled HA was found not only to penetrate the skin of nude mice and humans, but also to aid the transport of diclofenac to the epidermis (Brown et al. 1999). HA also has been reported to produce similar effects with ibuprofen (Brown et al. 2002; Brown and Martin 2001), clindamycin phosphate (Amr 2000), and cyclosporin (Brown and Moore 1996; Nazir et al. 2001).

FIG. 2 The effect of 1% w/w polysaccharide on the percentage of applied diclofenac (1.75% w/w) delivered to the skin, 48 hr after application of the formulation (n = 4, mean ± standard deviation, *p < .001 compared with control).

Ophthalmic Drug Delivery

Topically applied drugs into the eye generally have low bioavailability with only a small fraction of the instilled dose remaining on the target tissue of the eye (Jarvinen et al. 1995). The precorneal clearance of the drugs is due to a rapid drainage, resulting in considerable loss of the drugs via the nasolachrymal duct, combined with conjunctiva absorption. After clearance, drugs can reach the circulation, resulting in systemic side effects (Sasaki et al. 1996). Therefore, it is desirable to prolong the ocular residence of drugs and consequently to enhance local bioavailability.

HA has been used as a vehicle for topical ophthalmic drugs due to its viscoelasticity and mucoadhesive capacity (Lapcik et al. 1998; Kaur and Smitha 2002). Numerous studies have reported that HA is capable of prolonging the precorneal residence and/or increasing the bioavailability of pilocarpine (Gurny et al. 1987; Camber and Edman 1989; Camber et al. 1987, 1994; Saettone et al. 1989, 1991, 1994; Bucolo and Mangiafico 1999; Bucolo et al. 1998), tropicamide (Saettone et al. 1989; Herrero-Vanrell et al. 2000), timolol (Bucolo et al. 1998), gentimycin (Moreira et al. 1991a, 1991b; Bernatchez et al. 1993), tobramycin (Gandolfi et al. 1992), arecaidine propargyl ester (Langer et al. 1997) and S-aceclidine (Langer et al. 1997) in solution form. The mucoadhesion together with the increased structure conferred by HA are thought to be primarily responsible for the prolonged precorneal residence of the drugs. HA binds to the layer of natural mucin on the corneal epithelium (Saettone et al. 1994, 1989) and its shear thinning behavior provides no resistance to blinking, and thus results in good patient compliance.

Nasal Delivery

HA has been used as a component of vehicles for the nasal delivery of small molecular drugs and peptides with a view to increasing bioavailability as a result of bioadhesion and/or penetration enhancement (Morimoto et al. 1991; Lim et al. 2000, 2002; Castellano and Mautone 2002). Castellano and Mautone (2002) found that HA could be incorporated into a topical formulation of xylometazoline that provided effective relief of nasal congestion. In addition, Morimoto et al. (1991) reported that the bioavailabilities of nasally administered vasopressin and 1-deamino-8-D-arginine vasopressin in rats were increased more than 2-fold and 1.6-fold, respectively, when HA was in the formulation relative to the bioavailability attained in the polymers absence. The increases conferred by HA were found to be molecular-weight dependent with the high molecular weight HA fractions (more than 300 kDaltons) promoting an increase in bioavailability while low molecular weight HA (55 kDaltons) had no effect. The authors attributed the increases in bioavailability to the penetration enhancement induced by HA since in vitro, the ciliary beat frequency of rabbit nasal mucosal membranes appeared not to be affected by the presence of HA in solution (Morimoto et al. 1991).

However, the incorporation of HA into microparticulate delivery systems confers mucoadhesive properties (Lim et al. 2000, 2002). HA-contained microspheres were found to be markedly more mucoadhesive than those of chitosan-based microparticulates. In addition, the adhesive forces appeared not to be affected by the presence of a model drug, gentamicin (Lim et al. 2000). The bioavailability of intranasally administered gentamycin using HA-based microspheres (23.3% relative to intravenous dosing), although significantly higher than that obtained using drug solution (1.1%) or drug particles (2.1%), was not as high as that using chitosan-based particles (31.4%). The highest bioavailability (42.9%) achieved via intranasally administered particles was obtained using HA/chitosan microspheres (Lim et al. 2002). Therefore, researchers assumed that HA mainly acted as a mucoadhesive agent whereas chitosan had a major penetrating enhancement function with the combination producing a synergistic effect on drug absorption.

Pulmonary Drug Delivery

Protein and peptide drugs generally are delivered parenterally. However, clinical use of these drugs requires repetitive injections, which makes noninvasive dosing more desirable. As a result, pulmonary delivery of proteins and peptides has been extensively investigated with a view to eliciting systemic therapeutic effects. HA has been included as a component of an inhaled formulation of insulin to enhance the absorption of the hormone and achieve a sustained release (Morimoto et al. 2001; Surendrakumar et al. 2003). Morimoto et al. (2001) found that the inclusion of HA at a low concentration (0.1% and 0.2% w/v) to an insulin solution reasulted in an enhanced absorption, after intratracheal administration to rats. Such enhancement appeared to be independent of the pH of the solution.

In another study, insulin was spray-dried alone or in the presence of HA, HA/zinc ions, or HA/hydroxypropyl cellulose. Subsequently, the release profiles of the resultant insulin from particles were evaluated by monitoring the glucose levels of male Beagle dogs after inhalation (Surendrakumar et al. 2003). The administration of spray-dried insulin in the presence of HA to conscious dogs produced a sustained release and increased absorption of the hormone compared with spray-dried insulin alone. In addition, with the inclusion of Zn^{2 +} or hydroxypropyl cellulose to HA-containing particles, the absorption and the mean residence time of insulin were found to increase to 2.5–5- and 7–9-fold, respectively, relative to that of pure insulin powders administered via the pulmonary route.

Parenteral Drug Delivery

The potential applicability of nonimmunogenic HA as drug carriers for parenteral delivery was recognized by Drobnik (1991). Drug targeting to a specific site, such as the lymphatic system might be possible by conjugating a drug with HA or entrapping the drug in the HA matrix. Alternatively, such methods might be used to achieve sustained release or prolong the retention of drug within the body. For example, taxol, an anticancer drug, has been chemically linked to HA. The taxol-HA conjugate was found to selectively target human breast, colon, and ovarian cancer cell lines in vitro (Luo and Prestwich 1999; Luo et al. 2000). The linkage between HA and taxol was carried out by conjugating the taxol 2′-OH via a succinate ester to adipic dihydrazide-modified HA. After receptor-mediated cellular uptake, the free drug was released from the conjugate as a result of hydrolysis to achieve a concentration that resulted in cytotoxicity in cancer cell lines (Luo et al. 2000).

In addition, Sakurai et al. (1997a) found that conjugation of manganese and superoxide dismutase (Sakurai et al. 1997b) to HA showed nonimmunogenicity and enhanced the antiinflammatory activity of the two proteins in mice. In another study, a sustained release of human recombinant insulin-like growth factor-I, from a HA matrix, was achieved (Prisell et al. 1992). In vitro data indicated that the diffusion rate of the peptide was markedly decreased as a function of HA concentration, possibly due to peptide-HA interactions and were confirmed in vivo.

Liposomal Drug Delivery

Liposomal drug delivery is reported to increase drug solubility, prolong the half-lives of entrapped drugs in the body, and provide protection of the drug within the lipid particle. When HA was linked to lipids, the resultant liposomes possessed improved function and stability, had target-specific cells, and acted as sustained-release carriers of drugs for therapy (Yerushalmi et al. 1994; Yerushalmi and Margalit 1998; Peer and Margalit 2000; Eliaz and Szoka 2001; Peer et al. 2003).

Peer and Margalit (2000) have investigated the physicochemical properties of HA-modified liposomes and found that they provided comparable or better efficiency of encapsulation of some model chemicals, relative to unmodified liposomes. In addition, the drug release profiles of either modified or unmodified liposomes appeared to be similar and to follow a single rate constant (Peer and Margalit 2000). A further study also suggested that HA modification could confer cryoprotection to freeze-dried unilamellar liposomes, inhibiting the formation of larger multilamellar liposomes, which compromises the encapsulating efficiency of liposomes, upon reconstitution (Peer et al. 2003). In contrast, the encapsulation efficiency was found to decrease by 25–60% in the unmodified liposomes. The mechanism by which HA conferred cryoprotection of unilamellar liposomes was attributed to HA being able to replace the hydrogen bonds that stabilize the structures of liposomes during freeze-drying (Peer et al. 2003).

HA-modified liposome-encapsulated doxorubicin, an anticancer drug, was found to specifically target-cancer cells that express CD 44 (Eliaz and Szoka 2001). In addition, encapsulation of the drug in cell targeting liposomes was shown to increase the biological activity of doxorubicin against B16F10 murine melanoma cell line 4-8-fold relative to that of the free drug in terms of the IC₅₀ values; the actual extent of the increase being dependent upon the duration of treatment (Eliaz and Szoka 2001). The targeting ability of HA-modified liposomes also was tested by Yerushalmi et al. (1994) who found that more than 87% of the model drug, epidermal growth factor, could be encapsulated in the HA-modified liposomes. The authors found that the modified liposomes specifically adhered to the monolayers of the A431 cell line, the in vivo designated targets for the therapy of wounds and burns (Yerushalmi et al. 1994). A further study showed that such adherence was relatively robust and could be retained during cell migration, proliferation, and death, and under fluid flow (Yerushalmi and Margalit 1998).

Implantable Drug Delivery

HA has been utilized to develop an implantable delivery device with a view to conferring long-term delivery of protein drugs (Surini et al. 2003a, 2003b). HA has been reported to interact with chitosan and form a chitosan-HA complex, which in turn affects the drug release rate mainly due to the swelling effect that follows water penetration (Takayama et al. 1990). Surini et al. (2003b) produced a chitosan and HA implant containing insulin that enabled programmed release profiles to be achieved. The release of insulin from the system appeared to be zero order in nature, and the release rate was dictated by the quantity of formulation applied and the formulation composition, i.e., the chitosan-to-HA-mixing ratios and the amount of insulin loaded. For example, the release rate constant from a 50-mg pellet containing a chitosan-to-HA-mass ratio of 3:7 was found to be three times faster compared with that from a 135-mg pellet with a chitosan-to-HA-mass ratio of 16:84.

Gene Delivery

Recently, HA has been used to prepare DNA-HA matrices and microspheres with a view to achieving sustained gene delivery (Kim et al. 2003; Yun et al. 2004). In the matrices and microspheres, DNA entrapped in HA networks was cross-linked with adipic dihydrazide during preparation. These researchers found that the release kinetics of plasmid DNA from the matrices and microspheres could be controlled by optimizing the amount of DNA loaded into the matrices and the process for cross-linking. The release of DNA was shown to be initially dominated by the burst effect, followed by the erosion of HA matrix that could be designed to occur over the longer term (over 2 months). The DNA entrapped in the HA matrix was stabilized against enzymatic degredation but once released was found to be capable of transfection in vitro. In addition, Yun et al. (2004) found that DNA presented in HA microspheres transfected skeletal muscles in vivo. Moreover, after conjugation with monoclonal antibodies, it was found that DNA targetted specific cellular receptors, and the conjugates would distinguish between E- and P-selectin expressing cells. A summary of the drug delivery applications of HA is shown in Table 2.

TABLE 2 Summary of the drug delivery applications of HA

Route	Justification	Therapeutic agents	Publications
Ophthalmic	Increased ocular residence of drug that can lead to increased bioavailability	Pilocarpine, tropicamide, timolol, gentimycin, tobramycin, arecaidine polyester, (S) aceclidine	Jarvinen et al. 1995; Sasaki et al. 1996; Gurny et al. 1987; Camber et al. 1987, 1989; Saettone et al. 1994, 1991; Bucolo et al. 1998, 1999; Herrero-Vanrell et al. 2000; Moreira et al. 1991a, 1991b; Bernatchez et al. 1993, Gandolfi et al. 1992; Langer et al. 1997
Nasal	Bioadhesion resulting in increased bioavailability	Xylometazoline, vasopressin, gentamycin	Morimoto et al. 1991; Lim et al. 2002.
Pulmonary	Absorption enhancer and dissolution rate modification	Insulin	Morimoto et al. 2001; Surendrakumar et al. 2003.
Parenteral	Drug carrier and facilitator of liposomal entrapment	Taxol, superoxide desmutase, human recombinant insulin-like growth factor, doxorubicin	Drobnik 1991; Sakurai et al. 1997; Luo et al. 1999, 2000; Prisell et al. 1992, Yerushalmi et al. 1994, 1998; Peer et al. 2000; Eliaz et al. 2001; Peer et al. 2003.
Implant	Dissolution rate modification	Insulin	Surini et al. 2003; Takayama et al. 1990
Gene	Dissolution rate modification and protection	Plasmid DNA/monclonal antibodies	Yun et al. 2004; Kim et al. 2003.

CONCLUSIONS

Since its first discovery in 1934, HA's unique viscoelasticity, hygroscopicity, biocompatibility, and nonimmunogenicity properties of hyaluronan have facilitated its use in a variety of medicinal and pharmaceutical applications. As more and more is understood about the macromolecule and how its physicochemical properties can be manipulated by chemical modification or simply controlling its molecular weight, these applications may become more diverse. However, such developments need to be accompanied by an increased knowledge of its cellular functions and how it can be produced, assayed, and controlled in a more cost-effective manner.