(W1030-04-22) Hispolon Cyclodextrin Complexes and Cyclodextrins-in-Liposomes for Enhanced In Vitro Anticancer Activity against Melanoma

Purpose: Hispolon is a yellow phenolic pigment isolated from a mushroom species Phellinus linteus (1), has been investigated, and found effective for anti-inflammatory, antioxidant, and anticancer properties; however, low solubility and poor bioavailability has limited its potential clinical transition. Liposomes are non-toxic lipid-based carriers containing a phospholipid bilayer zone separating two aqueous compartments. The space offered by the lipid bilayer and inner water compartment is generally limited, and small molecules like hispolon tend to rapidly leak out from the liposomes resulting in poor drug loading and entrapment (2). A combined approach of cyclodextrins and liposomes could be utilized to trap hydrophobic drug complexed within cyclodextrin inside liposomes (3).

Drug-cyclodextrin in Liposomes (DCL) has been an exciting approach to solubilize drugs inside an aqueous compartment to protect the drug until release (4). Cyclodextrins are cyclic oligosaccharides that dynamically accommodate a guest lipophilic molecule to enhance its solubility and stability. During DCL formation, the lipophilic drug is trapped in liposomes' inner hydrophilic core due to the higher tendency of cyclodextrin towards an aqueous core. This ultimately increases the liposome stability and controls the release in comparison to conventional liposomes (5). On the other hand, liposomes prevent the possible dissociation of cyclodextrin complexes by shielding it against the external aqueous environment. It also avoids the interference of drugs on lipophilic bilayer, maintaining the rigidity of formulation. Recent publications on the complexation of Sulfobutylether β-cyclodextrin (SBEβCD) to form an inclusion complex with hydrophobic drugs like gemcitabine have enhanced the water solubility and dissolution rate cytotoxic activity of the drug (6). Thus, we hypothesized that hispolon could be encapsulated into liposomes after complexation with cyclodextrins to preserve its activity and enhance its solubility and drug release. In this study, an inclusion complex of Hispolon-SBEβCD complex (HSC) was prepared and trapped inside the liposomes further to investigate the in-vitro anticancer activity against melanoma cell lines. The HSC in liposomes (HCL) was also investigated for improved stability and drug efficacy compared to solution and sterically stabilized control liposomes (SSL).

Methods: SBEβCD was selected as entrapping molecule based on preliminary phase solubility studies, job’s plot analysis, and molecular modeling after comparison with hydroxypropyl-βCD. The hispolon-SBEβCD complex (HSC) was prepared using the Freeze-drying technique. HSC formation was confirmed using various solid characterization techniques (DSC, FTIR, Proton-NMR, SEM)(Figure 1). Sterically stabilized liposomes were prepared by a thin-film hydration technique. Lipids were dissolved in chloroform at a lipid molar ratio of 9:5:1 (DSPC/Cholesterol/DSPE-mPEG-2000) and dried into thin film under a vacuum. The dry film was hydrated to prepare a blank liposomal suspension, loaded with hispolon (1:10 drug: lipid ratio) using a remote loading method. HCL was prepared similarly by thin-film hydration technique whereby the hydration was performed with HSC solution (1:10 ratio). The addition of SBEβCD solution during hydration resulted in in-process complexation of hispolon to give HSL (Hispolon-SBEβCD-liposome) formulation. All the liposomes were sized using Lipex^TM extruder and purified using dialysis before the drug loading and physicochemical characterization. The drug loading and in vitro release (by dialysis method) between these three formulations were compared. The in-vitro anticancer efficacy of all the prepared formulations was investigated against B16-BL6 melanoma cell lines using an MTT assay.

Results: The complexation with SBEβCD increased the solubility of hispolon by 15 folds, and the overall stability of Hispolon as HSC improved in physiological pH (PBS, pH 7.4). The combination approach of trapping the HCL showed a smaller mean diameter ( < 120 nm), a narrow polydispersity index ( < 0.1), improved drug encapsulation ( >90%) compared to the control hispolon liposomes prepared by film hydration technique (Freeze-thaw, extrusion and dialysis steps common for all formulations), as shown in Table 1. In-process complexation of hispolon with SBEβCD (HSL) was not significantly loading the drug inside the liposomes compared to HCL, as a significant amount of free drug cleared off during dialysis. The HCL formulation showed a sustained release compared to control liposomes and HSL formulations. The in-vitro anticancer activity on melanoma cell lines (B16BL6) of HSC and HCL was higher than conventional liposomes and hispolon solution(Figure 2).

Conclusion: In this study, we prepared and optimized the cyclodextrin complex of hispolon with improved solubility and dissolution rate. Following that, conventional liposomes and DCL were formulated and investigated for their in-vitro anti-melanoma activity. The findings suggest that HSC complexes and HCL liposomes stand out as a potential formulation approach for chemotherapeutic administration of hispolon and similar molecules.

References: 1. Toopmuang P, Khamchum C, Punsuvon V. Detection and confirmation of hispolon in the mushroom Phellinus linteus. J. Sci. Asia. 2014 Apr 1;40:141-4.
2. Chen J, Lu WL, Gu W, Lu SS, Chen ZP, Cai BC, Yang XX. Drug-in-cyclodextrin-in-liposomes: A promising delivery system for hydrophobic drugs. Expert opinion on drug delivery. 2014 Apr 1;11(4):565-77.
3. McCormack B, Gregoriadis G. Drugs-in-cyclodextrins-in liposomes: a novel concept in drug delivery. International journal of pharmaceutics. 1994 Dec 5;112(3):249-58.
4. Gharib R, Greige-Gerges H, Fourmentin S, Charcosset C, Auezova L. Liposomes incorporating cyclodextrin–drug inclusion complexes: Current state of knowledge. Carbohydrate polymers. 2015 Sep 20;129:175-86.
5. Jain SK, Gupta Y, Jain A, Bhola M. Multivesicular liposomes bearing celecoxib-β-cyclodextrin complex for transdermal delivery. Drug delivery. 2007 Jan 1;14(6):327-35.
6. Rescifina A, Surdo E, Cardile V, Avola R, Graziano AC, Stancanelli R, Tommasini S, Pistarà V, Ventura CA. Gemcitabine anticancer activity enhancement by water soluble celecoxib/sulfobutyl ether-β-cyclodextrin inclusion complex. Carbohydrate polymers. 2019 Feb 15;206:792-800.


Figure 1. Compatibility screening of cyclodextrins and solid-state characterization of HSC complexes: (A) Phase solubility comparison between hispolon complexes with HPβCD and SBEβCD (B) Molecular modeling of HSC complexes (C) NMR comparison (D) Thermal analysis with DSC (E)FTIR analysis (F) SEM micrographs of complex formed


Table 1. Physicochemical characterization of liposomal preparations


Figure 2. In vitro anti-melanoma activity of investigated liposomal formulations