Polymer-tetrodotoxin conjugates to induce prolonged duration local anesthesia with minimal toxicity

Abstract

There is clinical and scientific interest in developing local anesthetics with prolonged durations of effect from single injections. The need for such is highlighted by the current opioid epidemic. Site 1 sodium channel blockers such as tetrodotoxin (TTX) are extremely potent, and can provide very long nerve blocks but the duration is limited by the associated systemic toxicity. Here we report a system where slow release of TTX conjugated to a biocompatible and biodegradable polymer, poly(triol dicarboxylic acid)-co-poly(ethylene glycol) (TDP), is achieved by hydrolysis of ester linkages. Nerve block by the released TTX is enhanced by administration in a carrier with chemical permeation enhancer (CPE) properties. TTX release can be adjusted by tuning the hydrophilicity of the TDP polymer backbone. In vivo, 1.0-80.0 µg of TTX released from these polymers produced a range of durations of nerve block, from several hours to 3 days, with minimal systemic or local toxicity.

Fig. 1

Design of a sustained release system for prolonged duration local anesthesia. a A polymer–TTX conjugate, designed to have a large TTX content with slow release, is placed near a nerve. Flux of TTX into the nerve is enhanced by a delivery system that acts as a chemical permeation enhancer. The polymer–TTX conjugate, schematized here as a hyperbranched structure has a much higher TTX loading than those actually produced here (which have 0.008 or 0.03 TTX molecules on each polymer chain). b Steglich esterification to synthesize TDP and TDP–TTX. The first step is optional, depending on whether the PEG moiety needs to be incorporated into the polymer

Fig. 2

Characterization of the TD/TDP polymers and their TTX conjugates. a ¹H-NMR spectra of the TD and TDP polymers. b Effect of polymer hydrophilicity (f_phil) on contact angle of the polymer thin film. c Effect of f_phil on half time of mass loss. d Degradation profiles of the TD and TDP polymers. e FTIR spectra of the T_gD₈ polymer and T_gD₈–TTX_H conjugates. The guanidinium group peak is characteristic of TTX. f Total ion chromatograms of a TTX standard (10 µg mL⁻¹) in citrate buffer and release media from T_gD₈–TTX conjugates. Both show a single peak at ~5.0 min. g Mass spectra confirm that the molecular weight of the peak observed at ~5.0 min corresponds to that of TTX (m/z 320.1 is [TTX + H]⁺). h Release profiles of 10 µg of free TTX or TTX from TD–TTX and TDP–TTX conjugates with differing f_phil. i Release half-time of 10 µg of conjugated TTX as a function of f_phil of polymer. In panels (a–d), (h), and (i), the open box denotes T_gD₈. Data in graphs are means ± SD, n = 4

Fig. 3

Peripheral nerve blockade with free TTX and T_gD₈–TTX conjugate. Effects of TTX dose on a frequency of successful blocks, b duration of sensory nerve blocks, c frequency of nerve blocks in the uninjected (contralateral) extremity, and d animal mortality. In all panels, the open box denotes T_gD₈–TTX, the gray box denotes T_gD₈–TTX_H. Data are means ± SD, n = 4

Fig. 4

Representative fluorescence images of sections of sciatic nerves and surrounding tissues. Tissues were harvested 1 and 4 h after injection of 0.25 mg of fluorescein sodium in 0.5 mL of PEG200 or PBS. Arrows indicate fluorescein sodium (green). Blue: cell nuclei stained with 4′,6-diamidino-2- phenylindole (DAPI). Data are representative of 4 animals in each group. Scale bars, 100 µm

Fig. 5

Retention in tissue of fluorescently labeled T_gD₈ injected at the sciatic nerve. a Representative fluorescent confocal photomicrographs 24, 48, and 168 h after sciatic nerve injection of 25 mg of FITC–T_gD₈ in 0.5 mL of PEG200. Green: FITC–T_gD₈; blue: cell nuclei (stained with DAPI). Scale bars, 200 µm. b Whole body imaging of fluorescence injection of 25 mg of CY5.5–T_gD₈ in 0.5 mL of PEG200. Fluorescence intensity is represented as radiant efficiency. c Quantification of data in panel (b), Data are means ± SD (n = 4). d Representative photographs of dissection of injection site 24 h after injection. Data are representative of 4 animals in each group

Fig. 6

Effects of f_phil (%) on efficacy and toxicity of sciatic nerve block. a Frequency of successful blocks, b duration of sensory nerve blocks, c frequency of nerve blocks in the uninjected (contralateral) extremity, and d animal mortality. Animals were injected with 1 or 10 µg of conjugated TTX in TD–TTX/PEG200 or TDP–TTX/PEG200. Data are means ± SD, n = 4

Fig. 7

Tissue reaction to materials. a, c Representative photographs of the site of injection upon dissection 4 and 14 days after injection of 25 mg of T_gD₈–TTX_H in 0.5 mL of PEG200. b, d Representative hematoxylin–eosin stained sections of muscles and adjacent loose connective tissue 4 and 14 days after injection of 25 mg of T_gD₈–TTX_H in 0.5 mL of PEG200. Scale bars, 100 µm. e–j Toluidine blue stained sections of nerve after injection of formulated T_gD₈–TTX_H. Scale bars, 50 µm. e, f Minimal peripheral injury (area enclosed by white dotted lines) seen in 1 of 3 nerves 4 days after injection of 25 mg of T_gD₈–TTX_H in 0.5 mL of PEG200. g–j Representative toluidine blue stained sections of nerve 4 days (g, h) and 14 days (i, j) after injection of 25 mg of T_gD₈–TTX_H in 0.5 mL mixture of PEG200 and PPG4000 (5/95, v/v), showing no injury. Data are representative of 3 animals in each group