Hyaluronan coated liposomes as the intravenous platform for delivery of imatinib mesylate in MDR colon cancer

Abstract

Imatinib mesylate has been evaluated for possible potential in treatment of colon cancer in recent times. However, due to significant reporting of P-gp expression in colon cancer, it can come across set back due to MDR. Therefore, in present work the liposomal formulation containing imatinib-bile salt conjugate was developed and investigated for its comparative performance in MDR colon cancer cells and surface modified with hyaluronic acid for achieving low hemotoxicity with stealth characteristics. Imatinib was successfully conjugated with sodium-deoxycholate by charged conjugation and evaluated through FTIR, DSC and PXRD. The developed conjugate (IM-SD) was encapsulated in liposomes and the conditions were optimized by Box–Behnken statistical design to achieve a size of 56.56 ± 1.23 nm along with 99.11 ± 0.89% entrapment efficiency (LIPO). The liposomes were surface modified with hyaluronic acid and the size was enhanced to 159.14 ± 3.2 nm (HA-LIPO). Flow cytometric studies demonstrated the enhanced uptake of P-gp substrate rhodamine dye in P-gp positive colo 320 colon cancer cells. In addition, an enhanced cellular internalization of HA-LIPO in CD-44 positive HT-29 and colo 320 cells indicates the targeting attributes of the hyaluronan coated liposomes. Finally, the hyaluronan coated liposomes were also found to have low opsonization activity.

Introduction

Imatinib mesylate or STI571 is a potent tyrosine kinase inhibitor with approved indication in chronic myloid leukemia (CML) [1] and gastrointestinal stromal tumors (GIST) [2]. In recent time it has been investigated for other tumors such as colon carcinoma [3]. However, imatinib mesylate is a substrate of P-glycoprotein (P-gp) and thus its present use in CML as well its future prospective in the treatment of colon cancer can be severely affected [4], [5], [6]. P-gp is a trans-membrane receptor that actively efflux out the substrate molecules like imatinib and reduce their intracellular levels. The sub-therapeutic intra cellular level of these xenobiotics either demands the increase in dose level or lead to treatment failure. In recent times a number of strategies were examined to modulate or moderate the P-gp action to re-sensitize the resistant cancer cells for the P-gp substrate drugs [7]. However, among various approaches for P-gp modulation, a novel drug delivery system such as nanoparticles provides the most lucrative choice to restore the therapeutic benefits of these molecules [8].

Liposomes are the closed structures of phospholipids formed by their spontaneous arrangement in aqueous media. Liposomal vesicular systems are generally of spherical shape with internal aqueous sac bounded by phospholipid bilayer [9]. Although liposomes are known to accommodate a wide range of drug from both lipophilic and hydrophilic categories, lipophilic moieties always have higher encapsulation potential [10], [11], [12]. Furthermore, higher lipophilicity ensures slow and sustained release of the drug from the liposomes without premature release [11], [13]. Therefore, imatinib was converted to the lipophilic conjugate by ionic complexation with sodium deoxycholate. Such strategies were successfully adopted with other bioactive hydrophilic molecules to increase their encapsulation in nanoparticles and sustaining their drug release profile [14], [15]. Furthermore, sodium deoxycholate has P-gp modulation potential that further justifies the conjugation of P-gp substrate molecule with P-gp modulator complexing agent [16]. Hence, imatinib was converted to a lipophilic bile acid conjugate before encapsulation into liposomes.

In present work we developed the cationic liposomes by using CTAB and coated them with hyaluronic acid by charged based surface conjugation [17]. Since, liposome has been proved to be clinically efficient drug delivery systems with low toxicity and industrial applicability [18], [19], [20] we focused on the formulations of this system to carry imatinib mesylate in MDR cancer cells. Furthermore phospholipids had proven inhibitory activity against P-gp [21], [22]. Therefore, this system was purposed to have anti-MDR potential via P-gp modulation which was investigated by flow cytometric cell uptake studies in P-gp negative as well as P-gp positive colon cancer cells. Additionally, to provide a non-toxic, hemo-compatible and low opsonization characteristics, the liposomes were surface modified with hyaluronic acid bio-macromolecule. Hyaluronic acid is a glycosaminoglycan (GaG) and is reported to induce hemocopatibility as well as stealth characters to the particulate systems [23], [24], [25]. Thus in present study, the surface modified liposomes were prepared to provide a hemocompatible, anti-MDR, and stealth intravenous platform to imatinib mesylate.