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. 2023 Jul 24:14:1214145.
doi: 10.3389/fphar.2023.1214145. eCollection 2023.

Ultrasound-assisted intravesical botulinum toxin A delivery attenuates acetic acid-induced bladder hyperactivity in rats

Qinggang Liu  1   2   3   4   5 , Yi Gao  2   5   6 , Huiling Cong  2   5   6 , Limin Liao  2   1   3   4   5   6
Affiliations

Ultrasound-assisted intravesical botulinum toxin A delivery attenuates acetic acid-induced bladder hyperactivity in rats

Qinggang Liu et al. Front Pharmacol. .

Abstract

Background: Intradetrusor injection of botulinum toxin A (BTX-A) is an effective treatment for overactive bladder (OAB). However, the occurrence of adverse events associated with BTX-A injection therapy hinders its acceptance among patients and its clinical promotion. Intravesical instillation of BTX-A offers a promising alternative to injection therapy for treating OAB. Nevertheless, due to the presence of the bladder permeability barrier (BPB) and the high molecular weight of BTX-A, direct instillation is unable to penetrate the bladder urothelium. Purpose: This study aims to investigate the safety and feasibility of ultrasound-assisted intravesical delivery of BTX-A and its potential benefits in a rat model of bladder hyperactivity induced by acetic acid instillation. Methods: Hengli BTX-A and microbubbles (MB) were mixed and prepared as a novel complex. The size distribution and zeta potentials of the complex were measured. On day 1, rats' bladders were instilled with 1 mL of saline, BTX-A (20 U in 1 mL), MB, or MB-BTX-A (20 U in 1 mL) complex with or without ultrasound (US) exposure (1 MHz, 1.5 W/cm2, 50% duty cycle, sonication for 10 s with a 10-s pause for a total of 10 min). The instillations were maintained for 30 min. After 7 days, cystometry was performed by filling the bladder with saline and 0.3% acetic acid (AA). Bladders were collected, weighed, and processed for immunoblotting, enzyme-linked immunosorbent assay (ELISA), histologic, and immunofluorescence analyses. Expression and distribution of SNAP-25 and SNAP-23 were assessed using Western blot and immunofluorescence. Calcitonin gene-related peptide (CGRP) in the bladder was detected using ELISA. Results: Intercontraction intervals (ICI) decreased by 72.99%, 76.16%, and 73.96% in rats pretreated with saline, BTX-A, and US + MB, respectively. However, rats treated with US + MB + BTX-A showed a significantly reduced response to AA instillation (57.31% decrease in ICI) without affecting amplitude, baseline pressure, or threshold pressure. Rats treated with US + MB + BTX-A exhibited increased cleavage of SNAP-25 and CGRP expression compared to the control group. Conclusion: Ultrasound-assisted intravesical delivery of BTX-A, with the assistance of MB cavitation, led to cleavage of SNAP-25, inhibition of calcitonin gene-related peptide release from afferent nerve terminals, and amelioration of acetic acid-induced bladder hyperactivity. These results support ultrasound-assisted intravesical delivery as an efficient non-injection method for administering BTX-A.

Keywords: bladder hyperactivity; botulinum toxin A; intravesical delivery; microbubbles; ultrasound.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XJ declared a shared parent affiliation with the authors QL and LL to the handling editor at the time of review.

Figures

FIGURE 1
FIGURE 1
Characteristics of microbubbles and ultrasound exposure. (A) Microbubbles (MB) were prepared according to manufacturer’s instructions. (B) Representative transmission electron microscope image of MB. (C) Hydrodynamic diameter distribution and zeta potential for MB. (D) Diagram of ultrasound (US) exposure.
FIGURE 2
FIGURE 2
Biocompatibility to bladder tissue of microbubbles with and without ultrasound in vivo. (A) Sprague-Dawley rats underwent intravesical instillation of saline or microbubbles with and without ultrasound 30 min. Bladder and body weights in each group. Bars represent mean ± SD. (B) Hematoxylin and eosin staining of bladders samples. (C) Expression of ZO-1 and E-cadherin analyzed using Western blot. Bars represent mean ± SD. (D) Immunofluorescence staining of E-cadherin protein in bladder samples.
FIGURE 3
FIGURE 3
Representative cystometrogram tracings after saline, botulinum toxin A, and/or microbubbles instillation with and without ultrasound. (A) Representative tracings of intraurethral cystometrograms (CMG) were recorded in urethane-anesthetized rats. CMG was performed in saline, botulinum toxin A (BTX-A)-, US + MB-, and US + MB + BTX-A–pretreated rats. (B) Bar plots represent the CMG parameters of amplitude, pressure threshold, pressure baseline, and intercontraction intervals between groups. Bars represent mean ± SD. ****, p < 0.0001 by one-way analysis of variance.
FIGURE 4
FIGURE 4
SNAP 23, cleaved and uncleaved SNAP-25, and CGRP expression in bladder tissue. (A) After cystometry, rats in each group were euthanized, and bladders were collected for subsequent Western blotting and immunofluorescence staining. Proteins levels of SNAP 23, cleaved and uncleaved SNAP-25 in bladder tissues of each group. (B) Representative immunofluorescence staining of SNAP 23 in bladder samples with or without ultrasound or microbubbles treatment. Scale bar = 50 μm. (C) Quantitative level of CGRP expression in bladder homogenates between groups using ELISA. Bars represent mean ± SD. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by one-way analysis of variance.
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