Abstract:

Objective To develop a carbon nanoparticle-manganese dioxide (CN-MnO₂) injection integrating visually identifiable staining and magnetic resonance (MR) T₁/T₂ dual-modal imaging, and to systematically evaluate its physicochemical properties, critical quality attributes, biocompatibility, and efficacy for gastrointestinal marking and localization. Methods CN-MnO₂ was prepared using a mild liquid-phase in situ redox deposition strategy and characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential analysis, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). The pH of CN-MnO₂ was directly measured using a pH meter. The osmolality pressure of CN-MnO₂ was determined by the freezing point depression method. The bacterial endotoxin was detected using the gel-clot Limulus amebocyte lysate test, which did not rely on absorbance readings. The appearance of the CN-MnO₂ injection solution stored at 4 ℃ under light shielding and sealed for 0, 5, and 10 days was observed, and its hydrated particle size, polydispersity index, zeta potential, and pH were also measured. With gadopentetate dimeglumine (Gd-DTPA) as the positive control, different concentration gradients were established, and T₁ and T₂ relaxation times were measured to construct concentration-dependent plots of the longitudinal and transverse relaxation rates (R₁ and R₂) and calculate the corresponding relaxivities (r₁ and r₂), compare relaxivity performance, and determine the working concentration for subsequent in vivo experiments. Cytocompatibility was assessed using the methyl thiazolyl tetrazolium (MTT) assay and live/dead staining. Sixteen male 6-week-old SD rats were randomly assigned to an experimental group and a control group (n=8 per group) and subcutaneously injected with CN-MnO₂ injection at a total Mn concentration of 0.80 mmol/L or an equal volume of normal saline, respectively. Short-term in vivo safety was evaluated by regular body weight monitoring, hematoxylin-eosin (HE) staining of major organs, hematological and serum biochemical analyses, inflammatory cytokine detection, and inductively coupled plasma optical emission spectrometry (ICP-OES) analysis. Eight male 5–6-month-old Bama minipigs were randomly assigned to a CN-MnO₂ group (n=6) and a carbon nanoparticle suspension injection (CNSI) group (n=2). CN-MnO₂ injection or CNSI was injected subserosally into the stomach, small intestine, and colon. Within 5 min after each injection, the maximum staining diameter at each injection site was measured and recorded. MR T1WI/T2WI scans were performed on days 3 and 7 after injection to evaluate MR visualization at the injection sites. At the end of observation, the injection sites and major organs were collected for histopathological evaluation by hematoxylin-eosin staining. Results CN-MnO₂ appeared as a black suspension, with no obvious aggregation or sedimentation after standing at room temperature (20-25 ℃) for 15 days. Its average hydrodynamic diameter was approximately (228.7±15.3) nm, the polydispersity index was 0.184±0.021, and the zeta potential was (-24.6±2.8) mV. XPS showed that CN-MnO₂ was mainly composed of C, O, and Mn, with oxygen accounting for approximately 68.82%. The Mn 2p spectrum exhibited two characteristic peaks at 642.4 and 653.1 eV. XRD indicated that low-crystallinity δ-MnO₂ was dispersed within the carbon matrix. The pH was 6.82±0.06, the osmolality was (298.4±6.7) mOsm/kg, and the bacterial endotoxin level was below the detection limit. In vitro MR imaging showed a clear concentration-dependent response, with r₁ and r₂ values of 9.6 L·mmol^-1·s^-1 and 17.2 L·mmol^-1·s^-1, respectively, indicating favorable T₁/T₂ contrast performance superior to that of Gd-DTPA. At 0.80 mmol/L, CN-MnO₂ showed no obvious cytotoxicity in vitro. Short-term safety evaluation in SD rats revealed no obvious inflammatory response, impairment of liver or renal function, or abnormal manganese accumulation in major organs. In Bama minipigs, the mean staining diameters of the CN-MnO₂ group in the stomach, small intestine, and colon were (0.65±0.12) cm, (0.61±0.11) cm, and (0.58±0.10) cm, respectively, with no significant difference among the three injection sites (P>0.05). The overall mean staining diameter was smaller in the CN-MnO₂ group than in the CNSI group (P<0.05). MR examinations showed that the injection sites were identifiable on both day 3 and day 7, with stronger signals observed on day 3. At the end of observation, no obvious pathological abnormalities were observed at the injection sites or in major organs. Conclusion A CN-MnO₂ injection with uniform particle size and stable physicochemical properties can be successfully prepared. This formulation integrates visible gastrointestinal staining with MR T₁/T₂ dual-modal imaging, providing a new strategy for preoperative gastrointestinal marking and localization as well as precise intraoperative resection.

Key words: Carbon nanoparticle-manganese dioxide, T₁/T₂ dual-modal imaging, Gastrointestinal surgery, SD rats, Bama minipigs