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To evaluate the effect of liposomes prepared from various natural and synthetic lipids in a rat bladder injury model in the absence or the presence of cholesterol and to elucidate the key structural elements necessary for the efficacy of liposomes required for alleviating bladder hyperactivity.The intravesical pressure was recorded using a transurethral catheter in adult female Sprague-Dawley rats anaesthetized with urethane (1.0 g/kg subcutaneously). Continuous cystometrograms (CMGs) were obtained by slowly filling the bladder with solutions of varying compositions after obtaining a baseline CMG with saline. Rat urothelium was injured with protamine sulphate (PS) and irritated by subsequent infusion of KCl (500 mM) for 1 h. Thereafter, liposomes prepared in KCl using several natural and synthetic phospholipids were infused for 2 h. The percentage reduction in bladder contraction frequency (BCF) was used as a comparative variable for judging the activity of different phospholipids.Exposure of rat bladder to sequential infusion of PS and KCl increased its BCF and empty liposomes of uncharged zwitterionic phospholipids markedly attenuated the PS-induced irritation and decreased the raised BCF. But empty liposomes prepared with either cationic or anionic charged lipids were not able to achieve the same effect. Addition of cholesterol did not significantly increase their efficacy. Optimal efficacy of liposomes was achieved with phosphatidylcholines with longer acyl chains and saturation in only one of the two acyl chains.These in vivo studies show that phospholipids attenuate the bladder irritation from KCl after PS-induced bladder injury.