5.4.1. Liposome Formulation of onaBoNTA

Liposomes are spherical lipid vesicles composed of phospholipid bilayers surrounded by an aqueous core. Liposomes can incorporate pharmaceutical drugs, both hydrophilic and hydrophobic, and transfer various sizes of drug molecules through the urothelium via the endocytosis mechanism [34]. Moreover, liposomes can coat the urothelium and assist in its repair in case of injury. Thus, only empty liposome bladder instillation can ameliorate the urinary symptoms and pain scale of patients with IC/PBS [35].

Chuang et al. examined the effects of liposome formulation of onaBoNTA (hereafter, lipotoxin) in a rat model [36]. The results demonstrated that intravesical lipotoxin instillation could cleave SNAP-25, inhibit calcitonin gene-related peptide release from afferents, and suppress bladder hyperactivity induced by acetic acid. Furthermore, Kuo et al. reported that bladder instillation of lipotoxin could reduce the urinary frequency at 1 month in OAB patients [37]. In a subsequent clinical trial, Chuang et al. recruited 62 participants with OAB and demonstrated that a single intravesical instillation of lipotoxin did work at 4 weeks by improving urgency but not beyond that time point [38].

Researchers are also considering the application of lipotoxin for IC/BPS or other bladder sensory disorders. However, Chuang and Kuo demonstrated that the therapeutic effect of a single intravesical instillation of lipotoxin for patients with IC/PBS would be similar to the placebo effect [39]. Recently, Lee et al. investigated the effects of lipotoxin on ketamine-induced ulcerative cystitis in rats [40]. The results showed repeated lipotoxin bladder instillation could protect the urothelium against the insults of urinary ketamine and its metabolites and restore the urothelial tight junction and adhesion proteins (i.e., zonula occludens-1 and E-cadherin). The chemodenervation effect of onaBoNTA could also be observed in this experiment, including the modulation of the detrusor M2 receptor, suppression of the neurogenic inflammation processes, and reduction in immune reactions. Hence, further studies

validating the long-term effects of lipotoxin in single or multiple instillations in treating sensory bladder disorders are warranted.

### 5.4.2. Intravesical Thermosensitive Hydrogel

Thermosensitive hydrogel consists of PEG-PLGA-PEG triblock copolymers, which can provide a composition to slowly release low-molecular-weight hydrophobic drugs lasting for 2 months [41]. The thermosensitive hydrogel exists in a liquid state at room temperature (25 ◦C) or below, but converts to a semisolid state at elevated temperatures, such as body temperature (37 ◦C) [42]. After the hydrogel is instilled into the bladder, it can act as a matrix filled drug for maintaining a prolonged exposure of drugs at the urothelium.

For treating patients with idiopathic OAB, Krhut et al. conducted a 1-month clinical trial using onaBoNTA embedded in an inert hydrogel [43]. The results revealed that intravesical instillation of 200U onaBoNTA, embedded in TC-3 gel, might have a therapeutic benefit for some patients with OAB. In a pilot study, Rappaport et al. used onaBoNTA embedded in TC-3 gel to treat patients with IC/PBS and reported a borderline therapeutic effect for participants on the visual analog scale at week 12 [44].

#### 5.4.3. Hyaluronan-Phosphatidylethanolamine

Hyaluronic acid, also called hyaluronan (HA), is a hydrophilic polysaccharide and a common ingredient in skincare products for its epidermis-healing function. A nonparticulate formula was developed by linking HA to phosphatidylethanolamine (PE), which increases HA levels through epidermal cell layers. This high viscosity formulation of HA-PE could be applied as a carrier for transferring large proteins, such as onaBoNTA, through the urothelium. In a rat model, Shatoury et al. proved that bladder instillation of onaBoNTA enmeshed in HA-PE could transfer onaBoNTA through bladder mucosa. According to the study, both routes of HA-PE and intradetrusor injections had comparable SNAP-25 cleavage [45].

### **6. Conclusions**

In this review, the suitable modalities for transferring onaBoNTA into the bladder were discussed. The summary of therapeutic effects in clinical trials among various drug delivery systems is illustrated in Table 1. The efficacy of current solutions did not reach a similar efficacy as intradetrusor injections. In sum, passive diffusion is less effective and practical in clinical use. Protamine sulfate failed to deliver the onaBoNTA into the detrusor layer of the bladder, even in an animal model. Using EMDA to conduct onaBoNTA into the bladder seems promising in treating neurogenic OAB in children. It resulted in a 9-month decrease of detrusor pressure and alleviated urinary incontinence episodes and vesicoureteral reflux. LESWs can help the onaBoNTA penetrate into the bladder and maintain its therapeutic effects for 2 months in a clinical trial. Using DMSO as a delivery agent of onaBoNTA may benefit patients with idiopathic detrusor overactivity by improving urinary symptoms and quality of life for 1–3 months. The concept of liposome-encapsulated onaBoNTA has been proven in rat models. However, in clinical trials, the current lipotoxin regimen can only improve the OAB symptoms of patients for 1 month. At present, a thermosensitive hydrogel is also under clinical trial for treating urothelial cancer by embedding mitomycin. In terms of treating OAB or IC/PBS, the therapeutic effect of thermosensitive hydrogel embedded with onaBoNTA is barely satisfactory. HA-PE, a novel agent, can carry onaBoNTA across the urothelium of rats' bladders. Additional clinical trials are needed to prove its efficacy. It is worth noting that most of the literatures in this review did not report serious adverse effects during the studies. It may attribute to the small sample size and single arm studies thus that determining causes and unexpected adverse events might be unnoticed. The needle-free delivery techniques of onaBoNTA through bladder instillation should continuously be improved for treating sensory bladder disorders, and the safety and efficacy are worthy of being further investigated.


**Table 1.** Clincal trials for sensory bladder disorders with onabotulinumtoxinA injection-free procedures.

MMC, myelomeningocele; DO, detrusor overactivity; OSS, O'Leary-Sant symptom scores; IC/BPS, interstitial cystitis/bladder pain syndrome; ICSI, ICPI, Interstitial cystitis symptom, and problem indices; VAS, visual analog scale; PPBC, Patient Perception of Bladder Condition; OAB-q, Overactive Bladder Questionnaire; FBC, functional bladder capacity; GRA, global response assessment.

**Author Contributions:** P.-Y.C. and W.-C.L. wrote the manuscript and figures, H.-J.W. collected information and references; Y.-C.C. supervised and revised the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported by Grants MOST104-2314-B-182A-081 from the Ministry of Science and Technology of the Republic of China and CMRPG8F0051-53 and CMRPG8J0271 from Chang Gung Medical Foundation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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*Review*
