Microfluidic fabrication of X-ray-visible sodium hyaluronate microspheres for embolization

Abstract

Catheter embolization is a minimally invasive technique that relies on embolic agents and is now widely used to treat various high-prevalence medical diseases. Embolic agents usually need to be combined with exogenous contrasts to visualize the embolotherapy process. However, the exogenous contrasts are quite simply washed away by blood flow, making it impossible to monitor the embolized location. To solve this problem, a series of sodium hyaluronate (SH) loaded with bismuth sulfide (Bi₂S₃) nanorods (NRs) microspheres (Bi₂S₃@SH) were prepared in this study by using 1,4-butaneglycol diglycidyl ether (BDDE) as a crosslinker through single-step microfluidics. Bi₂S₃@SH-1 microspheres showed the best performance among other prepared microspheres. The fabricated microspheres had uniform size and good dispersibility. Furthermore, the introduction of Bi₂S₃ NRs synthesized by a hydrothermal method as Computed Tomography (CT) contrast agents improved the mechanical properties of Bi₂S₃@SH-1 microspheres and endowed the microspheres with excellent X-ray impermeability. The blood compatibility and cytotoxicity test showed that the Bi₂S₃@SH-1 microspheres had good biocompatibility. In particular, the in vitro simulated embolization experiment results indicate that the Bi₂S₃@SH-1 microspheres had excellent embolization effect, especially for the small-sized blood vessels of 500-300 and 300 μm. The results showed the prepared Bi₂S₃@SH-1 microspheres have good biocompatibility and mechanical properties, as well as certain X-ray visibility and excellent embolization effects. We believe that the design and combination of this material has good guiding significance in the field of embolotherapy.

Fig. 1. Characterizations of Bi₂S₃ NRs. (A) TEM image and histogram of the size distribution. (B) XRD spectra. (C) In vitro CT images of Bi₂S₃ NRs with different concentrations (2.5, 5, 10, 15, 30, and 60 mg mL⁻¹, from left to right, respectively).

Fig. 2. SH and Bi₂S₃@SH microspheres fabrication schematic: (A) a schematic depicting imaginable (sodium hyaluronate) SH gel fabrication. (B) Schematic depicting microspheres fabrication. (C) Local magnification image of a flow-focused droplet microfluidic chip. (D) Optical micrograph of hydrated SH microspheres.

Fig. 3. Characterizations of SH microspheres: (A–C) SEM images of SH microspheres at various magnifications. (D) Histogram of the size distribution of SH microspheres. (E) Optical micrograph of SH microspheres post-swelling in PBS after 24 h. (F) FT-IR spectra of SH powder, BDDE, and SH microspheres.

Fig. 4. Characterizations of Bi₂S₃@SH microspheres: (A–C) SEM images of the SH-0, Bi₂S₃@SH-1, and Bi₂S₃@SH-2 microspheres, respectively, scale bar: 200 μm. (D–F) The high magnifications of (A–C), scale bar: 200 μm. (G) The high magnification of a single Bi₂S₃@SH-1 microsphere, scale bar: 30 μm. (H) The schematic of (G) internal structure. (I–L) The EDS-mapping images of (G), scale bar: 30 μm.

Fig. 5. Characterizations of Bi₂S₃@SH-1 microspheres: (A) XRD spectra. (B) Storage (G′) and loss (G′′) moduli were recorded for samples. (C) TGA curves of Bi₂S₃ NRs, SH-0, and Bi₂S₃@SH-1 microspheres. (D) In vitro CT images and the radiopacity values of the samples (n = 5).

Fig. 6. In vitro biocompatibility: (A) the cell viability of HUVECs co-cultured with Bi₂S₃@SH-1 microsphere extracts at 20 mg mL⁻¹ for 24 h, 48 h, and 72 h, respectively (n = 6). (B) Confocal microscopy images of HUVECs incubated with Bi₂S₃@SH-1 microspheres after 1 and 3 days, scale bar: 200 μm. (C) Hemolysis test of Bi₂S₃@SH-1 microspheres, the insert digital photograph of rabbit carotid blood supernatant and solution after centrifugation of samples, data were shown as mean ± standard deviation, **p < 0.01. (D) Blood coagulation test of samples for a different period, scale bar: 1 cm.

Fig. 7. In vitro vascular model embolization: (A) the design image of the PMMA chip. (B) In vitro embolization model, scale bar: 1 cm. (C) Before embolization, scale bar: 1 cm. (D) After embolization, scale bar: 1 cm.