Tracking the distribution of 99mTc-labeled 35 kDa hyaluronan fragment (HA35) via oral administration in mice

Background. Experimental studies indicate that the 35 kDa fragment of hyaluronan (hyaluronic acid, HA), termed HA35, significantly enhances intestinal mucosal healing and barrier integrity in various gastrointestinal pathological conditions, such as inflammatory bowel disease (IBD) and bacterial infections. Recent preclinical evidence has shown that orally administered HA35 improves intestinal epithelial barrier function, modulates inflammatory responses, reduces pathogen-induced intestinal damage, and interacts with specific cell surface receptors to exert protective effects. Despite these advancements, the pharmacokinetics, distribution, and precise mechanisms underlying the mucosal healing effects of HA35 on the gastrointestinal tract remain incompletely understood. Our prior animal studies demonstrated 99mTc-labeled HA35 ([99mTc]Tc-HA35) imaging effectively visualized lymphatic circulation in skin, subcutaneous tissues, and lymph nodes. Building upon these results, the current study employed [99mTc]Tc-HA35 imaging to track the absorption and distribution of orally administered HA fragments and to elucidate gastrointestinal lymphatics and lymphatic function in healthy mice.

Methods. Radiolabeling of HA35 with 99mTc was carried out at 80°C for 90 minutes. During this process, 99mTc pertechnetate [TcO₄⁻] was reduced by stannous chloride (SnCl₂) to Tc(V), subsequently binding to the carboxyl groups of HA35 to form a stable [99mTc]Tc-HA35 complex. Imaging studies were conducted in ten healthy BALB/c mice. [99mTc]Tc-HA35 (~9.25 MBq) was administered orally via gavage to five mice, while another five mice received intradermal injections of [99mTc]Tc-HA35 into all four paws to visualize lymphatic circulation and lymph nodes. Static scintillation images were acquired for 5–10 minutes at various intervals up to 18 hours post-administration. Approximately one hour before the final imaging session, 1.5% Evans Blue dye in saline was intradermally injected into the four paws of the mice to facilitate identification of lymph nodes corresponding to areas of radiotracer uptake. Following the final imaging, mice were euthanized for ex vivo biodistribution analysis.

Results. Using the direct radiolabeling protocol, [99mTc]Tc-HA35 was successfully produced with a radiochemical yield exceeding 95%, displaying good stability for at least 18 hours. After oral administration, predominent [99mTc]Tc-HA35 radioactivity was observed in the stomach and intestines, as well as in liver and spleen. Notably, radiotracer accumulation was detected in several lymph nodes, including mesenteric, lumbar, and iliac nodes, Persistent retention of [99mTc]Tc-HA35 was observed in lymph nodes, spleen, liver, and the gastrointestinal tract up to 18 hours post-administration, with moderate accumulation noted in the bladder. Following intradermal injection into the footpads, the lymphatic drainage patterns originating from distal extremities were visualized, demonstrating rapid radiotracer uptake in multiple lymph node groups, including the popliteal, inguinal, axillary, brachial, mandibular, lumbar, iliac, sacral, pancreaticoduodenal, and cervical nodes.