The effect of hepatopancreas homogenate of the Red king crab on HA-based filler

Determination of hyaluronidase activity by the turbidimetric method

Turbidimetric analysis was conducted according to previously developed protocols, with minor changes (Dorfman & Ott, 1948; Rapport, Meyer & Linker, 1950; Tam & Chan, 1983). The content of the fillers was diluted by phosphate buffer (160 mM disodium phosphate and 39 mM sodium chloride; adjusted to pH 5.55 with hydrochloric acid) to a concentration of 2.5 mg/mL HA. An aliquot of the hyaluronidase solution was added to 0.2 mL of the HA solution, and the reaction mixture was thoroughly mixed and incubated at 37 °C. At certain time points during incubation, 10–35 μL aliquots were taken and then immediately mixed with phosphate buffer (to 0.5 mL) and 2.5 mL of acidic albumin (24 mM sodium acetate, 79 mM acetic acid, 50 mM sodium chloride, 1% albumin, pH 3.5). The mixture was thoroughly mixed and incubated at room temperature for 50 min. The absorbance was then measured at 600 nm on a Hitachi U-2000 spectrophotometer.

Preparation of the homogenate of HPC

The HPC homogenate was prepared as follows. The Red king crab Paralithodes camtschaticus was caught by a crab catching vessel from the company CJSC Arcticservice in the Barents Sea during crab fishing season. The hepatopancreases of several crabs were separated and immediately frozen, stored, and transported at −25 °C. After the hepatopancreas was thawed at room temperature in laboratory conditions, distilled water and ice were added at a weight ratio of 1:8:2, and the mixture was stirred at a low speed for 60 min. The final mixture was placed in a separating column and left for 2 h at −14 °C. The aqueous phase of the homogenate was filtered using a hollow fiber ultrafiltration module “AP-3-300” (Scientific-Production Complex (SPC) Biotest, Russia). The color of the sterile solution was dark amber. This solution was concentrated using a hollow fiber ultrafiltration module “AP-3-1” (SPC Biotest, Russia). The protein concentration in obtained hepatopancreas homogenate samples was in the range of 2–3.5 mg/ml. An aliquot of the final (concentrated) product was stored at −20 °C. Its protein concentration was determined by a colorimetric method using the biuret reagent (Lovrien & Matulis, 1995). Albumin (lot 0205C125; Amresco, Fountain Pkwy, OH, USA) served as the calibration protein.

Purification of the HPC homogenate

The precipitation procedure was carried out at 4 °C. Ammonium sulfate (up to 50% saturation) was added to 1 mL of the homogenate to remove impurities from the obtained preparation. Ammonium sulfate was slowly added while stirring. After centrifugation for 10 min at 10,000×g and 4 °C, the precipitate was dissolved in 0.5 mL of phosphate buffer and dialyzed in the same buffer for 24 h at 4 °C. The obtained sample was centrifuged for 10 min at 10,000×g and 4 °C and then passed through a 0.22 μm filter (Millipore, Burlington, MA, USA). The obtained solution was colorless. The sample was analyzed by gel electrophoresis according to Laemmli (1970).

Atomic force microscopy

The samples were prepared for AFM as follows. Buffer solution was used to bring the filler concentration to 2.5 mg/mL. Before their use, filler samples were incubated for 40 min at 37 °C. For hydrolysates, an equal volume of the sample was taken from the reaction mixture after 5, 40 and 120 min of incubation at 37 °C. We also analyzed the purified sample of the HPC homogenate diluted 21 times by phosphate buffer after it was incubated for 40 min at 37 °C. Next, two μL of the sample was transferred to freshly cleaved mica and incubated for 5 min. The sample was then washed twice in a drop of distilled water (deionized by a type I Milli-Q system) for 30 s and air-dried. AFM imaging was performed with an AFM Ntegra-Vita microscope (NT-MDT, Russia) in noncontact (tapping) mode in air. The typical scan rate was 0.5–1 Hz. Measurements were carried out using cantilevers NSG03 with a resonance frequency of 47–150 kHz, ensuring a 10 nm tip curvature radius. The processing and presentation of the AFM images were performed using Nova software (NT-MDT, Russia) and Gwyddion 2.44 software (http://gwyddion.net/, Czech Republic).

Nuclear magnetic resonance

One-dimensional (1D) ¹H-NMR spectra were acquired with a Bruker Avance III 600 spectrometer (The Core Facilities Centre of Institute of Theoretical and Experimental Biophysics of the RAS) operating at a frequency of 600 MHz (¹H) and a probe temperature of 310 K. The samples were placed into NMR tubes with a 5 mm diameter. Standard pulse sequences from the Bruker NMR pulse sequence library were used in the experiments. The 1D-pulse sequence ZGPR was applied for the suppression of proton signals from water. The free induction decay was collected into 96 K data points using an acquisition time of 3.42 s. The spectrum width was 24 ppm, the 90° pulse width was 16 μs, and the relaxation delay was 10 s. The NMR spectra were obtained sequentially with time fixation.

Hyaluronic acid from rooster comb or filler content was diluted with phosphate buffer (160 mM disodium phosphate, 39 mM sodium chloride, pH 5.55) to a concentration of 2.5 mg/mL HA. D₂O (30 μL) and 30 μL of the purified sample of the HPC homogenate were added to 600 μL of HA solution preheated at 37 °C for 1 h. The sample was stirred, centrifuged for 1 min at 15,000×g, and placed into an NMR tube. The time points started with the addition of the HPC homogenate. After 50 spectra, the sample was transferred to an Eppendorf tube, and 10 μL of 4 mM 3-trimethylsilyl (2,2,3,3-2H4) propionate (TSP) in D₂O was added to the sample as a standard marker. The mixture was placed into an NMR tube, and the ¹H spectra were recorded. The control sample (600 μL of phosphate buffer, 30 μL of D₂O and 30 μL of the decontaminated HPC homogenate preparation) did not produce any signals, except for ¹H of H₂O (Fig. S1). The Bruker TOPSPIN program was used to process the spectra and calculate integrals. The concentrations of HA were calculated at each time point on the basis of the proton signals of the acetyl group of the flexible or compact parts of the HA chains. The concentration was calculated relative to the known concentration of the added TSP marker. The molar concentration of HA was estimated from its disaccharide units, namely, d-glucuronic acid and N-acetyl-d-glucosamine.

The exponential curve of Figure “Hydrolysis of Hyaluform HA by purified HPC homogenate: (b) accumulation kinetics of the ¹H signal of the N-acetyl-d-glucosamine acetyl group of HA chains (integral under the peaks)” was approximated, as described by $$C=1.2096+2.347\ast (1-\mathrm{e}\mathrm{x}\mathrm{p}(-0.876\ast x))+1.8997\ast (1-\mathrm{e}\mathrm{x}\mathrm{p}(-0.02724\ast x))$$

The exponential curve of Figure “Hydrolysis of HA from rooster comb by purified HPC homogenate: (b) accumulation kinetics of the ¹H signal of the N-acetyl-d-glucosamine acetyl group of HA chains (integral under the peaks)” was approximated, as described by $$C=1.8559+1.3706\ast (1-\mathrm{e}\mathrm{x}\mathrm{p}(-2.1222\ast x))+1.7783\ast (1-\mathrm{e}\mathrm{x}\mathrm{p}(-0.2954\ast x))$$

Turbidimetric analysis of HA-based fillers

The turbidimetric method was used to determine hyaluronidase activity. In contrast to the method for measuring the HA solution viscosity, the turbidimetric method is less laborious and requires a smaller amount of substrate. This method is based on two properties of high molecular weight HA: it binds to albumin in acidic conditions, and it forms aggregates. Light scattering by the aggregates was determined by using the dynamic light scattering method or measuring the optical density of the sample. As soon as the high molecular weight forms of HA are transformed into low molecular weight forms (as a result of hydrolysis), aggregates can no longer form. This phenomenon underlies the determination of hyaluronidase activity by this method and forms the basis for kinetic studies. It has been implicitly shown that HA aggregates no longer form with acidic albumin when the average molecular weight of HA is 6–8 kDa (Rapport, Meyer & Linker, 1950).

Figure 1A demonstrates the concentration dependence of the optical density on the amount of HA registered by turbidimetric analysis. It is important to note that unmodified HA obtained from rooster comb binds to albumin much more effectively than HA from the other sources. In further experiments, the optical density values were considered to be correct if they were on a linear part of these curves. Figure 1B shows the hydrolysis of the native HA from rooster comb. The decreased ability of HA to form aggregates during incubation with the homogenate was used as an indicator of HA cleavage.

Turbidimetric analysis of the filler HA hydrolysates resulting from the homogenate of the Red king crab hepatopancreas (HPC) produced an unexpected result (Fig. 1C). Instead of the anticipated reduced capacity of HA to form aggregates with acidic albumin, the opposite effect was observed in the first 40 min: the hydrolysis products of the filler HA bound to the protein and formed aggregates with it more effectively (i.e., the optical density increased). After 40 min of the reaction, the optical density in the turbidimetric samples decreased. The figure also demonstrates a sufficient level of reproducibility in the turbidimetric data. The same results for the hydrolyses of HA from Hyaluform (Fig. 1D) and Revofil Ultra (Fig. S2) were obtained with the commercially available hyaluronidases Lydase and Liporase.

The dependence of the hydrolytic efficiency on the total HPC homogenate protein in the reaction mixture was also investigated (see Fig. 1E). An increase in enzyme concentration resulted in the hydrolysis of HA into fragments that were unable to bind rapidly with acidic albumin. At a ratio of 1:1 (by weight) of total HPC homogenate protein to HA, little aggregation was detected after 10 min of hydrolysis, indicating that almost all HA molecules were converted into their short forms within 10 min. With a decrease in enzyme concentration in the reaction mixture, the conversion of HA to these short forms was slower. The most effective binding of acidic albumin to HA occurred (i) 10 min after the start of hydrolysis at a ratio of protein to HA of 1:5 and (ii) 20 min after the start of hydrolysis at a ratio of protein to HA of 1:10. Thus, a ratio of protein to HA of 1:10 or 1:15 was chosen as optimal for the experiments.

The hydrolysis reaction was stopped by increasing the temperature of the reaction mixture to 100 °C for 10 min: after 40 min of incubation at 37 °C, the reaction mixture (in a tightly closed Eppendorf tube) was placed in a boiling water bath for 10 min, after which the incubation was continued at room temperature (Fig. S3). This simple approach will make it easy to obtain partially hydrolyzed cross-linked HA in future studies (see “Discussion”).

AFM of HA-based fillers

Studying the HA hydrolysates using AFM required the purification of the HPC homogenate sample to remove pigments and contaminants (see “Materials and Methods”). The level of hyaluronidase activity of the purified homogenate was the same as that of the initial sample (Fig. 1F), and SDS-PAGE protein electrophoresis did not reveal significant differences in the sets of proteins between these two samples of the preparation (Fig. S4).

The AFM images of the HPC homogenate diluted 21 times by phosphate buffer show the presence of amorphous structures, but the decontaminated homogenate resembles “globular”-like structures with a height of 4–30 nm (Fig. S5). These are most likely complexes of proteolytic and other enzymes.

Since the filler HA solutions were stored at 4 °C, the samples were first warmed at 37 °C for 40 min before AFM analysis. The heat treatment did not significantly affect the results of the turbidimetric analysis (Fig. S6). The AFM images of the Hyaluform filler show spherical-like structures from several to 300 nm in height (Fig. 2).

The population of these spherical-like structures can be divided into three groups according to their size and quantity. The first group includes a small population of structures 150–300 nm in height (Fig. 2A). Similar structures are observed in the Revofil Ultra filler preparation (Fig. S7). The second group is represented by a large population of structures 10–20 nm in height. An even larger population of structures are several nanometers in height and form the third group; these structures are formed as a dense layer that surrounds the other structures mentioned above (Fig. 2B).

The AFM method was used to analyze the hydrolysates of HA from the Hyaluform filler. The hydrolysates were obtained with the decontaminated HPC homogenate. Aliquots were taken from the reaction mixture at 5, 40 and 120 min of incubation and transferred to mica for AFM studies (Fig. 3; Fig. S8).

The AFM results show small gaps, consisting of spherical-like structures, on the sample surface (Fig. 3A) at the 5 min time point of hydrolysis. After 40 min of hydrolysis, these gaps are expanded (Fig. 3B). After 120 min, the spherical-like structures almost disappear, and filamentous mesh structures appear (Fig. 3C). The filamentous mesh structures are similar to those in the AFM images of non-cross-linked HA, as shown in (Jacoboni et al., 1999).

Furthermore, the AFM image of the reaction mixture after 5 min of incubation shows spherical-like structures about 600 nm in height or greater (Fig. S9). After 40 min of hydrolysis, there are fewer of these large structures, and many spherical-like structures up to 150 nm in height are observed. After 120 min, amorphous structures are detected in the samples, while structures up to 80 nm are practically undetectable.

The AFM analysis of native HA from rooster have shown the presence of an amorphous layer formed by aggregates up to 10 nm high, and a large number of globular-like aggregates up to 100–200 nm (Figs. S10 and S11). The hydrolysis of HA from rooster comb by purified HPC homogenate resulted in the disappearance of the most of large aggregates, the appearance of extended filaments and destruction of the amorphous layer with formation of various small aggregates (40 min of incubation). After 120 min of hydrolysis, the large aggregates almost completely disappeared and mostly small aggregates up to 8 nm high are present in the reaction mixture.

NMR of HA-based filler

The NMR spectrum of HA from Hyaluform reveals a weak ¹H signal of the sugar rings in the range of 5.9–3.3 ppm and a minor ¹H signal of the acetyl groups of N-acetyl-d-glucosamine at around 2 ppm (Fig. 4). The low detection levels of these signals are due to the close packing of the high molecular weight cross-linked molecules of HA in the filler.

The reaction mixture obtained from the hydrolysis of the Hyaluform filler by purified HPC homogenate was incubated in the NMR spectrometer at 37 °C for several days with NMR analysis at certain time points. Figure 4 shows a scan taken on the 5th day of sample incubation. The protons (¹H) of the NH group, sugar rings, and acetyl groups of N-acetyl-d-glucosamine are observed to have significantly higher signals compared with the initial HA. No significant proton signals of acetic acid, amino acids, or other compounds are detected in the spectrum (the presence of these signals would indicate bacterial contamination of the sample during the incubation). Figure 5A shows details of the changes in the proton spectra of the acetyl groups of N-acetyl-d-glucosamine during the experiments. The protons of the acetyl group of the flexible parts of the HA chains are represented by a symmetric, narrow signal in the NMR spectrum, while the protons of the same acetyl group of HA included in associations (the compact part of HA chains) are represented by a wide, asymmetric signal that shifted to a high field by 0.04 ppm. The asymmetry of the peak is due to the different sizes of HA associations in the solution.

The concentrations at each time point of the reaction were calculated from two proton signals of the HA acetyl group in both the flexible and compact parts of the HA chains (Fig. 5B). According to the NMR analysis, HA degradation in the reaction mixture lasts for several days, but the maximum reaction rate is observed in the 1st h of incubation. There are several mechanisms of hydrolysis: cross-link hydrolysis, sugar chain hydrolysis, and both reactions. All of these pathways decrease the size of HA associations in solution and thus increase their flexibility. This is confirmed by an increase in the corresponding signals in the proton NMR spectra; the integrated areas under the signals correspond to the apparent concentration.

NMR analysis of the hydrolysis reaction was performed for native HA from rooster comb (Fig. S12). The kinetics of the ¹H signal accumulation of the N-acetyl-d-glucosamine acetyl group during the hydrolysis reaction of native HA had the same pattern as that of filler HA (Fig. S13). Since native HA does not have cross-links, it can be affirmed that sugar chain hydrolysis occurred for filler HA.

The real concentration of HA from the filler prior to hydrolysis was calculated as the concentration of its disaccharide units (d-glucuronic acid and N-acetyl-d-glucosamine); this value was 6.89 mM without taking cross-links into account. According to the NMR data, the total apparent concentration of the acetyl groups from the flexible and compact parts of the HA chains was 5.92 mM at the end of the experiment. The NMR spectra also reveal that small sugars were not formed as a result of hydrolysis by the homogenate.