Liu, X. Michael*; Harmon, Patricia S.†; Maziarz, E. Peter‡; Rah, Marjorie J.§; Merchea, Mohinder M.§
Hyaluronan or hyaluronic acid (HA) is a naturally occurring, non-branched polysaccharide that consists of alternately repeating monosaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine linked by β 1–3 and β 1–4 glycosidic bonds. HA is present to some extent in virtually all biological fluids and tissues, including human tears.
HA is thought to possess anti-inflammatory properties,1–3 has been shown to play a role in corneal wound healing,4–6 and is thought to have a protective effect against oxidative damage to cells by inhibiting free radicals.2,7,8 The protective effect appears to increase with increasing HA concentrations.2 It is hypothesized that HA also increases corneal wettability due to the enhanced water retention on the corneal surface, making it useful in the treatment of dry eye.9 It is a natural lubricant, is extremely hygroscopic, enhances tear stability,10 and has been found to play a significant role in reducing protein deposition on contact lens materials.11 HA has become increasingly important to the ophthalmic industry due to its unique properties.11–13 For example, HA has been added to contact lens products to enhance the wettability and comfort of contact lenses.14 In addition, research has indicated that there is a correlation between the molecular weight of HA and its biocompatibility/biological functions in ocular tissues, osteoarthritis, and general biological research.12,15–20
Given the correlation between the molecular weight of HA and its biological functions, molecular weight should be an important consideration when developing ocular products; however, the molecular weights of HA used in ophthalmic products such as multipurpose solutions (MPS), eye drops, and contact lens packaging solutions vary. As such, the purpose of this research was to characterize and compare the molecular weights, molecular weight distributions, and concentrations of HA present in a series of commercially available HA-containing ophthalmic products.
Eleven commercially available HA-containing ophthalmic products, Biotrue MPS (Bausch + Lomb, Rochester, NY, USA), Sodyal MPS (Omisan Pharmaceuticals, Italy), Avizor Unica Sensitive MPS (Avizor, Spain), Hy-Care MPS (Sauflon Pharmaceuticals Ltd., UK), Hyalu-Sol MPS (Italy), Blink Contact Lubricating Eye Drops (Abbott Medical Optics, Santa Ana, CA, USA), Aquify Long-Lasting Comfort Drops (Ciba Vision, Duluth, GA, USA), Safigel contact lens packaging solution (Safilens, S.R.L., Staranzano, Italy), Weicon Brand B MPS (Weicon Optics Ltd., Shanghai, China), Eye Secret MPS (Hydron Optics Ltd. Taiwan), and Simply One MPS (Perret Opticiens, Carouge/Geneva, Switzerland) were obtained for use in this study. Aqueous size-exclusion chromatography with on-line triple detection (SEC-TD)21,22 was used to determine the molecular weights and concentrations of HA in these commercially available products, including marketed contact lens MPS and contact lens packaging solutions. An Integrated PL-GPC50 Plus instrument (Varian, Palo Alto, CA, USA) was used for the SEC-TD analysis. This instrument contains a pump for solvent delivery, an oven to house the columns, and three detectors: dual-angle (45 and 90 degrees) light scattering, differential refractometer, and viscometer. Upon eluting off the column, the sample entered the light scattering detector, after which the flow was split 1:1 between the refractometer and viscometer.
Two Ultrahydrogel Linear (Waters Corporation, Milford, MA, USA) columns connected in series were used to separate the HA from the rest of the components in the test products. These columns are packed with a semi-rigid polymeric gel that is hydroxylated polymethyl methacrylate in nature. They can be expected to have a small amount of free carboxyl groups as a result of hydrolysis, which can interact adversely with components present in the ophthalmic products (i.e., cationic disinfectants and preservatives). Using 0.1 M triethylamine hydrochloride with 1% acetic acid as the mobile phase minimizes interactions because the partially ionized triethylamine effectively caps the anionic sites on the column packing material.
The SEC-TD system was calibrated using a 0.5 mg/mL solution of a polyethylene oxide (PEO) narrow standard (Mp = 258,000 Da; Mw/Mn = 1.12) prepared in borate buffered saline. This was used for determining the interdetector delay constants, as well as for the detector constants. The dn/dc value of the aqueous solution of PEO used for calibration was 0.135 mL/g, as given on the certificate of analysis provided with the standard. Data acquisition and calculations were performed using Cirrus Multi GPC/SEC software version 3.1 (Agilent Technologies, Santa Clara, CA, USA).
In order to confirm the presence of HA and reduce the chemical interferences, we ran SEC-Triple Detection experiments of the samples before and after treatment with hyaluronidase. The schematic of the SEC-Triple Detection system is shown in Fig. 1. Two-sample t tests were used to compare the molecular weights of HA in each of the products with the molecular weight of HA in Biotrue MPS.
The following simplified equations reflect the correlations between the detector signals and polymer molecular weight, intrinsic viscosity, and molecular radii for conformation determination:
where RdRI, Rvisc, and RLS(θ) are signals of the refractive index detector, viscosity detector, and light scattering detector, respectively; KRI, Kvisc, and KLS are the instrument constants of refractive index, viscosity, and light scattering detectors, respectively. The concentration of the sample solution is denoted by C, and the specific refractive index increment is given by dn/dc. The viscosity and weight-average molecular weights are Mv and Mw, respectively. For an individual fraction from SEC, Mv is equal to Mw because they are treated as monodisperse samples. The intrinsic viscosity of the polymer is represented by IV. The scattering angle is θ and P(θ) is the form factor, which relates the angular variation in scattering intensity to the mean square radius of the particle. The excess light scattering intensity at the zero angle is calculated from the 90-degree excess scattering intensity by using the particle size information for P(θ) calculation under appropriate assumptions.23 The refractive index of the solvent is denoted as n0, λ0 is the wavelength of the light, and NA is the Avogadro number. Equation 4 is the Mark-Houwink equation, with KM and exponent a being Mark-Houwink constants. The value of exponent a (or the conformational coefficient) can also be used to predict the combined effect of polymeric chain conformations and the presence of branching. It is well known that values of a ≥ 1.0 indicate a rigid rod conformation, values between 0.5 and 0.8 indicate a random coil in a theta and good solvent, respectively, and a ≤ 0.5 indicates a spherical or branching conformation.24 Equations 5 and 6 are the formulae used for calculating the radius of gyration (Rg′) and hydrodynamic radius (Rh) which are derived from the Einstein viscosity law for particles in suspension under certain assumptions.21–23
SEC-TD reveals the presence of polymer materials without providing positive identification (i.e., chemical structure, end group chemistry, and molecular formula). Therefore, an orthogonal test was employed to confirm the presence of HA in the polymeric peak analyzed in each of the 11 ophthalmic products tested. Hyaluronidase is an enzyme known to digest or break down HA molecules specifically. Each of the 11 products was analyzed by SEC-TD both before and after exposure to an aqueous solution of hyaluronidase. In order to allow for complete reaction, the digestion experiments were allowed to sit overnight. If a peak in the chromatogram consisted of only HA, that peak would completely disappear after exposure to the enzyme. That is, it would move to the solvent front which is not monitored in this method. If the polymeric peak in the chromatogram of a given product had the same intensity before and after the hyaluronidase treatment, it was concluded that HA was not detectable in that product using the chromatographic method described in this paper. Finally, if the polymeric peak was present but with a decrease in intensity after treatment with the enzyme, it was concluded that there was at least one other component in the ophthalmic product co-eluting with HA. This interference hindered the ability to accurately determine the concentration and molecular weights of HA in this product in the same way used for the other products. The three scenarios described above were observed in Biotrue, Eye Secret, and Simply One, respectively (Figs. 2–4).
The average molecular weight values (Mw, Mn) and molecular weight distributions (PD) of HA in each of the 11 ophthalmic products tested were obtained by SEC-TD according to Equation 3. The values are given in Table 1. Each reported value is the mean result of a minimum of three measurements.
The molecular weight distribution of HA for one of the ophthalmic products tested (Safigel contact lens packaging solution) is shown in Fig. 5. The overlay of differential refractive index (dRI) chromatograms of three ophthalmic products (Blink Contact Lubricating Eye Drops, Sodyal MPS, and Safigel Contact Lens Packaging Solution) is shown in Fig. 6. These are three ophthalmic products with HA molecular weights ranging from 155,000 Daltons (Da) to 624,000 Da and HA concentrations ranging from 0.003 to 0.15%. An earlier retention time for the refractive index peak indicates a higher molecular weight. Refractive index peak area is directly correlated with the concentration of HA present in the sample.
The weight-average molecular weights (Mw) of the products tested ranged from 155,000 to 1,400,000 Da and the number-average molecular weight (Mn) of the products ranged from 99,000 to 927,000 Da. When the molecular weights of the solutions were compared, the molecular weights of HA in the other ophthalmic products reported were determined to be statistically lower than those of HA in Biotrue MPS (p < 0.05 for all comparisons). The polydispersity (PD = Mw/Mn) values of most of the samples tested were between 1.4 and 2.0, which is consistent with the PD of raw materials.21
The concentration of HA was determined by comparing the differential refractive index (dRI) detector response of the HA peak in each solution with the response of a solution containing 0.01% (w/w) HA. The dRI detector is considered to be a universal detection system in which HA with various molecular weights exhibits similar refractive index values. Therefore, the dRI response is directly proportional to the concentration of HA regardless of its molecular weight.
The concentration of HA in the 11 ophthalmic products tested ranged from 0.003 to 0.15%, as shown in Table 1. In general, the concentrations in both the contact lens packaging solution and eye drops tested were higher than those in the MPS. Both the concentrations and the average molecular weights of HA vary significantly in different MPS solutions. Each MPS solution contains cationic disinfectants/preservatives such as polyhexamethylene biguanide and/or quaternary amines (i.e., polyquaternium-1, PQ-1) for disinfecting contact lenses. It is well known that the anionic HA will interact with these cationic disinfectants. Therefore, it is reasonable to believe that there is a certain reaction equilibrium between HA and other cationic species for the product development process and product stability perspectives.
A wide range of HA molecular weights and concentrations were found in the 11 ophthalmic products characterized in this study, with the HA found in Biotrue MPS having the highest molecular weight (p < 0.05). Many ophthalmic product manufacturers use heat or autoclave sterilization in the manufacturing of their products. However, HA is degraded by high temperatures, and therefore the use of heat sterilization may result in ophthalmic products with lower molecular weight HA. The newly granted patent (Sterile HA solutions) describes the challenging and novel manufacturing process used to preserve the high molecular weight of HA during the sterilization process for Biotrue MPS.25
The categorization of the molecular weight of HA is important as higher molecular weight HA has been shown to exhibit many biological benefits12,15–17,19,20 while lower molecular weight HA has shown complex biological responses which are potentially less favorable to the eye.12 High molecular weight HA is believed to be larger than 800,000 Da.12,15–18 Any HA with a molecular weight less than 800,000 Da is considered to be medium or low molecular weight HA.
The biological and physical benefits of high molecular weight HA are reported to include protection of corneal16,17 and conjunctival epithelium,16 and anti-inflammation.16,20 Pauloin et al (2008) investigated the in vitro ability of three different molecular weights of HA (1,000,000, 100,000, and 20,000 Da) to reduce the preservative toxicity of benzalkonium chloride (BAK) on human epithelial models.17 Testing of two human epithelial cell lines showed that HA 1,000,000 Da significantly reduced all of the BAK-induced cytotoxic effects observed in the study. Specifically, HA 1,000,000 Da decreased oxidative stress, BAK-induced mitochondrial mass, chromatin condensation and plasma-membrane permeability, and DNA fragmentation of conjunctival cells. In addition, HA 1,000,000 Da protected the actin structure against BAK-induced cytoskeleton disorganization. The cytoprotective effects of HA observed in the study decreased with the decreasing molecular weight of HA. The authors concluded that only the high molecular weight HA had a significant cytoprotective effect.
Pauloin et al (2009) have also shown that high molecular weight HA had significant protective effects against UVB radiation.16 More specifically, their results indicated that high molecular weight HA significantly decreased UVB-induced cell death, significantly decreased UVB-induced caspases-3 and -8 activation but not caspase-9 activation, and significantly decreased UVB-induced IL-6 and IL-8 production. No protective effect was found for UVB-induced oxidative stress or UVB-induced DNA damage and p53 activation. The authors concluded that high molecular weight HA provided anti-inflammatory and anti-apoptotic signals to cells exposed to UVB radiation.
Guillaumie et al (2010) studied the influence of HA molecular weight on its water-binding capacity, rheological properties, precorneal residence time in rabbits, and the tolerance of ophthalmic solutions.18 The authors tested two types of hyaluronic acid: medium molecular weight HA from Bacillus fermentation (Mw = 680,000, 770,000, 890,000, or 1,140,000 Da) and high molecular weight HA from Streptococcus fermentation (Mw = 1,5000,000 or 2,250,000 Da). Their results showed that all HA samples bound very high amounts of water and the amount of bound water was independent of molecular weight and the origin of the HA. The kinematic viscosity was strongly dependent on the molecular weight and the concentration of HA. The kinematic viscosity of commercial eye drops typically varies from 2 to 7 centipoise18 which can be met using both medium and high molecular weight HA depending on the concentration of HA used. A prolonged residence time in the precorneal area was observed for a 0.3% solution of high molecular weight HA compared to a medium molecular weight solution at the same concentration. In vivo ocular tolerance was noted for both types of HA after topical installation onto the corneal surface. The authors noted that medium molecular weight HA is superior to high molecular weight HA due to the ease of sterilization by filtration and manufacturing of medium weight HA. However, techniques for sterilization of high molecular weight HA have been developed to overcome this concern.25
Kikuchi et al (1996) showed that when comparing medium (800,000 Da) and high (1,900,000 Da) molecular weight HA, the protection against cartilage degeneration in rabbits was significantly greater with the high molecular weight HA 2 weeks following knee surgery.15 Their results showed that high molecular weight HA is clinically efficacious in the treatment of incipient osteoarthritis. Hsieh et al (2008) have also shown the benefits of using high molecular weight HA for the treatment of osteoarthritis.19 In a comparison of high versus low molecular weight HA, their results showed effective protection for articular cartilage with the high molecular weight HA. This was evidenced by inhibition of MMP-2, MMP-9, u-PA, and PAI-1 expression. Finally, Huang et al (2010) revealed that high molecular weight HA (6,000,000 Da) was more effective in downregulating pro-inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α than lower molecular weight HA (500,000 to 730,000 Da).20 The above studies all reaffirm the benefits of higher molecular weight HA, including high viscoelasticity,12,18 protection of corneal16,17 and conjunctival epithelium,16 protection of articular cartilage, and anti-inflammation.16,20
Aqueous size-exclusion chromatography with on-line triple detection proves to be an effective tool to determine the concentrations of HA and to evaluate the molecular weights and molecular weight distributions of HA present in ophthalmic products. A wide range of HA molecular weights and concentrations were found in the 11 ophthalmic products characterized in this study, with the HA found in Biotrue MPS having the highest molecular weight (p < 0.05). The molecular weight of HA used in ophthalmic products may impact clinical performance. More research may be warranted to better understand the clinical and biological implications of the molecular weights of HA in ophthalmic products.
X. Michael Liu
Global Research & Development
Bausch + Lomb, Inc.
1400 N. Goodman St. Rochester, NY 14609
All authors are employees of Bausch + Lomb, Inc., Global Research & Development, Rochester, New York. The research was funded by Bausch + Lomb, Inc.
Received: January 30, 2013; accepted July 12, 2013.
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