Hyaluronidase, Bovine testes

Hyaluronidase, Bovine testes (Hyaluronate 4-glycanohydrolase; Hyaluronoglucosaminidase) is an endoglycosidase that depolymerizes Hyaluronic acid (HA) (HY-B0633A) by cleavage of glycosidic bonds. Hyaluronidase degrades HA and activates membrane receptors that trigger pathways converging in NF-κB activation. Hyaluronidase is employed in the research of granulomatous foreign body reactions, soft-tissue necrosis caused by vascular compromise and uncomplicated nodules, overcorrection, inflamed nodules or tissue ischemia associated with HA filler injection^{[1][2][3][4][5][6]}. In Vitro:1. Hyaluronidase inhibition assay^[3]
(1) Mix 10 μL of 8 mg/mL Hyaluronidase with the sample in a buffer solution (0.1 M acetate buffer, pH 3.6) in a test tube, and incubate at 37 °C for 20 minutes.
(2) Add 20 μL of 12.5 mM calcium chloride to the mixture and incubate at 37 °C for another 20 minutes.
(3) Treat the activated Ca²⁺ mixture with 50 μL of 2.4 μg/mL hyaluronic acid in the buffer, and incubate further at 37 °C for 40 minutes.
(4) Add 2 μL of 0.4 N sodium hydroxide and 20 μL of 0.4 N potassium tetraborate tetrahydrate, then incubate in a water bath at 100 °C for 3 minutes.
(5) After cooling to room temperature, add 600 μL of DMAB solution to the mixture and incubate at 37 °C for 20 minutes. (6) Measure the absorbance at 585 nm using an enzyme-linked immunosorbent assay (ELISA) reader.
In Vivo:1. The effect of hyaluronidase on skin necrosis in HA induced vascular complications^[7]
(1) Anesthetize 10 rabbits weighing 3-4 kg using intramuscular injections of ketamine (50 mg/kg) and xylazine (10 mg/kg).
(2) Shave the hair off the ear, then use electrocoagulation to seal the blood vessels behind the ear. Inject 1% lidocaine (HY-B0185) to numb the skin near the ear, and make an incision to expose the anterior branch of the posterior auricular artery.
(3) Under the surgical microscope, a 30-gauge needle was used to puncture the artery, and 0.25 mL of HA filler was injected. The small initial amount of HA filler injected into the artery can expand the arterial lumen, helping to position the needle within the artery.
(4) Inject HA filler into both ears, with one ear receiving a subcutaneous injection of 750 IU of Hyaluronidase. Five rabbits (4-hour intervention group) were injected with Hyaluronidase 4 hours after HA injection, while another five rabbits (24-hour intervention group) received Hyaluronidase 24 hours after the HA injection.
(5) Capture the progression of skin changes and measure the final necrotic areas. Use the Statistical Package for Social Sciences (SPSS) software for statistical analysis.
2. The effect of hyaluronidase on the anti-tumor activity based on nanoparticles ^[8]
(1) This experiment used female Balb/c mice. 4T1 cells were suspended in PBS at a density of 1×10⁶/50μL and were injected subcutaneously into the back of the mice either unilaterally or bilaterally.
(2) When the tumor volume reaches 110-150 mm³, in vivo experiments are conducted. Mice are randomly divided into 4 groups (n=5): saline injection group, Hyaluronidase injection group, NM-Ce6 induced PDT group, and Hyaluronidase + NM-Ce6 induced PDT group. In the Hyaluronidase injection group, 1500 U of Hyaluronidase is dissolved in 50 µL PBS and injected into each tumor. One hour after the Hyaluronidase injection, each mouse receives an intravenous injection of 200 µL NM-Ce6, with a Ce6 concentration of 1 mg/mL (Ce6 dose = 10 mg/kg). Twenty-four hours after the NM-Ce6 injection, the tumors are exposed to 660 nm light for 60 minutes at a power density of 2 mW/cm² to perform PDT.
(3) On the 6th day after the initial treatment, repeat the same procedure. Record tumor volume and mouse weight for a total of 12 days. After the treatment, remove 4 tumors from each group and weigh them, and remove the main organs from each group for H&E staining.