Characterization of the Molecular Weight of Hyaluronan in Eye Products Using a Novel Method of Size Exclusion High-Pressure Liquid Chromatography

Abstract

Purpose: Hyaluronan (HA) exists in two forms, high molecular weight HA (HMWHA) and low molecular weight HA (LMWHA), which have distinct physiological functions. Therefore it is imperative to know the form of HA within pharmaceutical products, including eye products. This study developed an accurate, sensitive, and quantitative method to characterize the form of HA in eye products. Thereafter, the effects of the HA-containing eye products on corneal wound healing were investigated.

Methods: The MW distributions and concentrations of HA in over the counter eye products were determined by size exclusion chromatography (SEC) high-pressure liquid chromatography (HPLC). The effects of the eye products containing HA on corneal wound healing were characterized both in vitro and in vivo using the scratch assay and the debridement wound model, respectively.

Results: The concentrations and MWs of HA were successfully determined within a range of 0.014 to 0.25 mg/mL using SEC HPLC. The concentrations of HA in the ophthalmic products varied from 0.14 to 4.0 mg/mL and the MWs varied from ∼100 kDa to >2500 kDa. All but one HA-containing eye product had an inhibitory effect on corneal wound healing, whereas pure HA promoted corneal wound healing.

Conclusions: A novel SEC-HPLC method was developed for quantifying and characterizing the MW of HA in eye products. Although HA promoted corneal wound healing, HA-containing eye products inhibited corneal wound healing, likely caused by preservatives.

Translational relevance: SEC-HPLC could be implemented as a routine method for determining the form of HA in commercially available ophthalmic products.

Figure 1.

Standardization of the chromatographic profile for HMWHA, LMWHA, and ULMWHA by size exclusion HPLC. HMWHA, mid-MWHA, and LMWHA were applied to a size exclusion HPLC at increasing concentrations starting from 0.0025 mg/mL to 2.5 mg/mL, and the concentration and molecular weight of HA were successfully determined within a range of 0.014 to 0.25 mg/mL. (A–C) Linear regression equations and r² were calculated for HMWHA (A), mid-MWHA (B) and LMWHA (C). (D–E) Chromatographic profiles for HMWHA, mid-range MWHA, and LMWHA at increasing concentrations, specifically 0.0025, 0.005, 0.025, 0.05, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, and 2.5 mg/mL, are shown.

Figure 2.

Chromatographic profile for HA in different HA-containing eye products by size exclusion HPLC. Eight HA-containing eye products were administered to size exclusion HPLC and monitored at 204 nm, and an overlay of their chromatographic profiles is shown.

Figure 3.

Identification of HA in HA-containing eye products by analyzing susceptibility to hyaluronidase (Hyalase) digestion. Eight HA-containing eye products were treated with Hyalase or not and then administered to size exclusion HPLC and monitored at 204 nm. An overlay of the chromatographic profiles of the HA-containing eye products that were treated with Hyalase or with enzyme buffer in the absence of Hyalase is presented.

Figure 4.

Characterization of the molecular weight of HA by agarose gel electrophoresis. HA standards and OTC eye products were subjected to agarose gel electrophoresis, and molecular weight was determined based on the electrophoretic profile of two HA ladders that contained HA standards of known molecular weights. The molecular weights of HA (kDa) in each ladder (I and II) are indicated next to each ladder.

Figure 5.

Effects of HA of different molecular weights and HA-containing OTC eye drops on corneal epithelial wound healing were investigated using a scratch assay. Confluent hTCEpi were subjected to scratch wounds and the cells allowed to migrate into the cell free area for 24 hours in presence of HMWHA, mid-MWHA, and LMWHA at a concentration of 0.05 mg/mL or various HA-containing OTC eye products diluted 1:10 in media. Images were captured at zero, six, 18, and 24 hours under an EVOS microscope or an LSM 800 confocal microscope (Carl Zeiss Microscopy LLC), and the cell-free area was calculated using the plugin in Image J designed by Suarez-Arnedo et al. that is specific for in vitro wound healing assays. The cell-free area was calculated at each timepoint, and the wounded area was calculated as a percentage compared to zero hours. (A) Indicates the migration profile of corneal epithelial cells in the presence of HMWHA, mid-MWHA, and LMWHA compared to PBS control. (B) Indicates the migration profile of corneal epithelial cells in the presence of HA-containing eye drops that contained HA primarily in the HMWHA range compared to the HMWHA standard. (C) Indicates the migration profile of corneal epithelial cells in the presence of HA-containing eye drops that contained HA primarily in the mid-MWHA range compared to the mid-MWHA standard. (D) Indicates the migration profile of corneal epithelial cells in the presence of HA-containing eye drops that contained HA primarily in the LMWHA range compared to the LMWHA standard.

Figure 6.

The effects of HA-containing OTC eye drops on corneal epithelial cell viability (A) and proliferation (B) was investigated using the CCK-8 assay. For such, corneal epithelial cells were treated with selected HA-containing OTC eye products at 1:10 and compared to HMWHA and PBS (vehicle control). Error bars: SD. Asterisk represents P ≤ 0.05.

Figure 7.

The effect of HA-containing eye drops on corneal epithelial wound healing in vivo. Effects of HA of different molecular weights and HA-containing OTC eye products on corneal epithelial wound healing were investigated in vivo using the debridement wounding model. For such, a 1 mm–diameter injury site was demarcated on the cornea of mice using a biopsy punch, and thereafter the corneal epithelial cells were removed using an AlgerBrush II rotating burr. Fluorescein stain was used to identify the injured area, and images were captured at zero, 12, and 24 hours. The injured area was calculated by manually drawing the wound edge as region of interest (ROI) using the polygonal selection function of the Image J software, and, thereafter, the area of the ROI was measured, and data were represented as the percentage of the wounded area remaining compared to zero hours. (A) Effects of HMWHA (2 mg/mL), mid-MWHA (2 mg/mL), mid-MWHAʹ (0.3 mg/mL), LMWHA (2 mg/mL), and ULMWHA (2 mg/mL) on corneal epithelial wound healing were investigated and compared to PBS, which served as the vehicle control. (B) Representative images of fluorescein-stained murine corneas that were quantified in panel A, imaged under a stereomicroscope at one, 12, 16, and 24 hours after injury. (C) Effects of eye products containing HA primarily in the HMWHA range compared to a HMWHA standard and PBS. (D) Representative images of fluorescein-stained murine corneas that were quantified in panel C, imaged under a stereomicroscope at one, 12, 16, and 24 hours after injury. (E) Effects of eye products containing HA primarily in the mid-MWHA range compared to a mid-MWHA standard at two different concentrations and PBS. (F) Representative images of fluorescein stained murine corneas that were quantified in panel E, imaged under a stereomicroscope at one, 12, 16, and 24 hours after injury.