International Journal of Pharmaceutics 461 (2014) 82–88 Contents lists available at ScienceDirect International Journal of Pharmaceutics jo ur nal ho me p ag e: www.elsevier.com/locate/ijpharm Probing influence of methodological variation on active loading of acetazolamide into nanoliposomes: Biophysical, in vitro, ex vivo, in vivo and rheological investigation Prashant B. Pisal a , Manjusha A. Joshi b , Mahesh N. Padamwar b , Sharvil S. Patil a , Varsha B. Pokharkar a,∗ a Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Department of Pharmaceutics, Erandwane, Pune 411038, Maharashtra, India b Sai Life Sciences Ltd, Pune 411057, Maharashtra, India a r t i c l e i n f o Article history: Received 23 August 2013 Received in revised form 14 November 2013 Accepted 18 November 2013 Available online 26 November 2013 Keywords: Gradient liposomes Intra-ocular pressure Glaucoma Encapsulation efficiency Acetazolamide a b s t r a c t In the present work comparative evaluation of acetate and pH gradient techniques for effective drug loading in liposomes has been investigated. The acetazolamide (ACZ) loaded liposomes prepared by two methods were analyzed by vesicle size analysis, zeta potential, percent encapsulation efficiency, in vitro drug release studies and intraocular pressure lowering activity. ICH guidelines were followed for determining stability of the prepared liposomes. The superiority of acetate gradient method for active loading of acetazolamide has been established. The prepared acetate gradient positive liposomes showed extended hypotensive effect when compared to other liposomal formulations. Thus ACZ loaded liposomes prepared by acetate gradient technique may serve as promising ocular delivery system in the treatment of glaucoma. The current work emphasizes the fact that the techniques used for active drug loading into liposomes strongly influence the pharmaceutical performance of the final formulation. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Liposomes, a vesicular drug carrier system has revolutionized the area of novel drug delivery system pertaining to their wide scope of applications. These carrier systems are self-aggregated bilayer spherical structure of lipid surrounding aqueous compart- ments (Lasic, 1991; Ulrich, 2002). Most of the formulation scientists have been fascinated by these bio-compatible carrier systems owing to their ability to incorporate both water soluble as well as water insoluble molecules, protect proteins, nucleic acids and small molecules from degradation (Torchilin, 2005). The pharma- ceutical performance of liposomes is governed by the encapsulation efficiency, vesicle size distribution and stability of the prepared dis- persion. Passive and active drug loading techniques are used for entrapment of drug molecules within liposomes. Reports indicate that active drug loading prove promising in achieving high entrap- ment efficiency of the liposomes (Nichols and Deamer, 1976; Haran et al., 1993). One of the hurdles toward development of liposo- mal dosage forms is poor drug entrapment. This may be attributed to the lack of comparative knowledge of different active loading ∗ Corresponding author. Tel.: +91 20 25437237; fax: +91 20 25439383. E-mail address: vbpokharkar@yahoo.co.in (V.B. Pokharkar). techniques and its influence on pharmaceutical performance of the prepared liposomes. ACZ, a carbonic anhydrase inhibitor (CAI), orally used for glau- coma, was selected as model drug molecule. ACZ may become a preferred choice of drug in its class if one can be able to reduce various side effects associated with it (Kaur et al., 2002). In order to overcome the side effects of orally delivered ACZ, topical deliv- ery of the same may prove beneficial. Further the topical delivery of ACZ often presents advantages such as dose reduction, faster onset of action, marked reduction in side effects with improved patient compliance. However the topical delivery of ACZ may be challenged with the factors such as solubility and permeability. The performance of ACZ by topical route has been improved by different approaches such as cyclodextrin complexation (Loftsson et al., 1994), use of high dose of ACZ at the site of action (Flach et al., 1984), formulation of ACZ into gel (Kaur et al., 2000) and suspension, preparation of novel drug delivery system including liposomes. The topical route for ACZ, formulated as liposomes has shown promising results in the reduction of increased IOP. How- ever, liposomes prepared by reverse phase evaporation and thin film hydration technique have shown low entrapment of ACZ (El- Gazayerly and Hikal, 1997; Hathout et al., 2007). It is believed that the percentage drug loading which depends on the method of preparation of liposomes would ultimately influence the ther- apeutic efficacy of the final formulation. Thus the objective of the 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.11.034