Injectable electrospun fiber-hydrogel composite sequentially releasing clonidine and ropivacaine for prolonged and walking regional analgesia

Abstract

Rationale: Peripheral nerve block is a traditional perioperative analgesic method for its precise pain control and low systemic toxicity. However, a single low dose of local anesthetic merely provides a few hours of analgesia, and high dose results in irreversible toxicity, whereas continuous infusion of anesthetics is expensive and complicated. Therefore, it is necessary to develop a long-acting and sensory-selective local anesthetic for safe perioperative analgesia. Methods: An injectable composite comprising ropivacaine-loaded poly (ε-caprolactone) electrospun fiber and clonidine-loaded F127 hydrogel (Fiber-Rop/Gel-Clo composite) was developed for long-acting and walking regional analgesia with barely one dose. The peripheral nerve blockade effect of the composite was evaluated in a rat sciatic nerve block model. Also, the biodegradability and biosafety of the composite was evaluated. Results: The preferentially released Clo from the hydrogel rapidly constricted the peripheral arterial vessels, reducing the blood absorption of Rop and thus enhancing the local Rop accumulation at the injection site. The subsequently sustainable release of Rop from the fiber, significantly prolonged the sciatic nerve block of rats. Remarkably, an amazing sensorimotor segregation effect was achieved, as the sensory blockade (32.0 ± 1.4 h) lasted significantly longer than the motor blockade (20.3 ± 0.9 h). Additionally, the Fiber-Rop/Gel-Clo composite presented good biodegradability and biosafety in vivo. Conclusions: Our designed Fiber-Rop/Gel-Clo composite with minimal invasion, prolonged synergistic analgesia, and strikingly sensorimotor segregation effect, posted a promising prospect for regional long-term walking analgesia in clinical treatment.

Keywords: clonidine; electrospun fiber; injectable hydrogel; long-acting regional analgesia; ropivacaine; sensorimotor separation.

Scheme 1

Schematic illustration of fabrication and sciatic nerve blockade effect of the Fiber-Rop/Gel-Clo composite. (A) The Fiber-Rop/Gel-Clo composite was fabricated by combining Rop-loaded electrospun PCL nanofiber and Clo-loaded F127 hydrogel. (B) The rat sciatic nerve blockade effect of the Fiber-Rop/Gel-Clo composite. (C) The mechanism of long-acting and walking regional analgesia in vivo. Created with BioRender.com.

Figure 1

Characterization of Fiber-Rop/Gel-Clo composite. (A) Scanning electron microscopy (SEM) images of the Rop-loaded fiber (Fiber-Rop), F127 hydrogel (Gel), and Fiber-Rop/Gel composite; Scale bar, 10 µm. (B) Fluorescence microscope of fluorescently labeled Rop-loaded fiber (FITC, green), Clo-loaded F127 hydrogel (RhB, red), and Fiber-Rop/Gel-Clo composite (Merge); scale bar, 100 µm. (C) Photographs showing F127 hydrogel and Fiber-Rop/Gel composite at 4 °C and 37 °C. (D) Rheological behaviors of the Clo-loaded F127 hydrogel and Fiber-Rop/Gel-Clo composite, measured at 1% strain and 1.0 Hz frequency with the temperature set from 0 °C to 40 °C at a speed of 2 °C/min. G′, storage modulus; G″, loss modulus. (E, F) In vitro cumulative release of Rop and Clo from the Fiber/Gel composite in phosphate buffered saline with or without lipase (20 U/mL), (E) 0-14 days, (F) 0-10 hours. Data are shown as mean ± standard error, (n = 3/group; *P < 0.05, **P < 0.01). (G) Photographs showing the Fiber-Rop/Gel-Clo composite with good injectability.

Figure 2

Evaluation of sciatic nerve blockade effect in rats. (A) Schematic diagram of rat sciatic nerve block. Created with BioRender.com. (B) Comparison of the sciatic nerve blockade effect in rats after different doses of free Rop solution or Fiber-Rop/Gel composite injection. Data are shown as mean ± standard error, (n = 3/group, n.s, not significant; **P < 0.01, ***P < 0.001). (C) Motor block was evaluated with a four-point rating scale. (D) Duration of motor block was the time from score 4 to score 2. (E) Sensory block was determined by paw withdrawal thermal latency (PWTL), and percentage of maximum possible effect (%MPE) was calculated as the effect of sensory blockade. (F) Duration of sensory block was the time from %MPE reaching 50% to less than 50%. Data are shown as mean ± standard error, (n = 6/group; n.s, not significant; **P < 0.01, ***P < 0.001).

Figure 3

Clo reduces the concentration of Rop absorbed by local vasoconstriction. (A) Schematic diagram of the experimental program to evaluate the role of Clo by ultrasound imaging. Created with BioRender.com. (B) Representative images of arterial diameter around the sciatic nerve at 0 h, 4 h, and 8 h after injection of Fiber-Rop/Gel composites with or without Clo. White arrows show the location of blood vessel and white circles show the outline of blood vessel. Scale bar, 1.0 mm. (C) Qualitative analyses of arterial diameter around the sciatic nerve at 0 h, 4 h, and 8 h after injection of Fiber-Rop/Gel composites with or without Clo. (D) Representative images of blood flow velocity at 0 h and 4 h after injection of Fiber-Rop/Gel-Clo composites. (E) Qualitative analyses of blood flow velocity around the sciatic nerve at 0 h, 4 h, and 8 h after injection of Fiber-Rop/Gel composites with or without Clo. (F) The dynamic pharmacokinetics of RhB released from Fiber-Rop-RhB/Gel composite and Fiber-Rop-RhB/Gel-Clo composite in rats. Data are shown as mean ± standard error, (n = 3/group; **P < 0.01, ***P < 0.001).

Figure 4

Assessment of biodegradation and biosafety of Fiber-Rop/Gel-Clo composite in rats. (A) In vivo degradation of Fiber/Gel composite. Local anatomical structures of sciatic nerves and surrounding muscles, and the potential composite remnants were captured on 7, 14, and 21 days after injection. Cyan arrows indicate sciatic nerves and red arrows indicate the composite remnants. Scale bar, 1 cm. (B) Hematoxylin and eosin (H&E) staining of sciatic nerves and surrounding muscles; scale bar, 500 μm. Toluidine blue O (TBO) staining of sciatic nerves; scale bar, 100 μm. Immunohistochemical analysis of inflammatory factors (TNF-α, IL-6) in the sciatic nerve and surrounding muscles; scale bar, 500 μm. TUNEL staining of sciatic nerves; scale bar, 100 μm. 'N' and 'M' represent the sciatic nerve and muscle, respectively.

Figure 5

Assessment of systemic toxicity of Fiber-Rop/Gel-Clo composite in rats. (A) H&E staining of the heart, liver, spleen, lung, kidney, and brain (hippocampus CA1); scale bar, 200 μm. (B) The serum levels of CK-MB, LDH, AST, ALT, Cr, and BUN among the seven groups. The light gray backgrounds indicate the upper normal limit.