Biomacromolecules 2015, 16, 3784−3791
Nanoassemblies Based on Supramolecular Complexes of Nonionic Amphiphilic Cyclodextrin and Sorafenib as Effective Weapons to Kill Human HCC Cells
Maria Luisa Bondì,a Angela Scala,b Giuseppe Sortino,c Erika Amore,d Chiara Botto,d Antonina Azzolina,e Daniele Balasus,e Melchiorre Cervello,e and Antonino Mazzaglia*,c
aCNR-ISMN Istituto per lo Studio dei Materiali Nanostrutturati – U.O.S. di Palermo, Via Ugo La Malfa 153, 90146 Palermo, Italy
bDipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università di Messina, V.le F. Stagno D’Alcontres 31,98166 Messina, Italy
cCNR-ISMN Istituto per lo Studio dei Materiali Nanostrutturati c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali dell’ Universitá di Messina, V.le F.Stagno D’Alcontres 31, 98166 Messina, Italy
dDipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Universitàdegli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
eIstituto di Biomedicina e Immunologia Molecolare “A. Monroy” – Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146 Palermo, Italy
Sorafenib (Sor), an effective chemiotherapeutic drug utilized against hepatocellular carcinoma (HCC), robustly interacts with nonionic amphiphilic cyclodextrin (aCD, SC6OH), forming, in aqueous solution, supramolecular complexes that behave as building blocks of highly water-dispersible colloidal nanoassemblies. SC6OH/Sor complex has been characterized by complementary spectroscopic techniques, such as UV−vis, steady-state fluorescence and anisotropy, resonance light scattering and 1H NMR. The spectroscopic evidences and experiments carried out in the presence of an adamantane derivative, which competes with drug for CD cavity, agree with the entrapment of Sor in aCD, pointing out the role of the aCD cavity in the interaction between drug and amphiphile. Nanoassemblies based on SC6OH/Sor display size of ∼200 nm, negative zeta-potential (ζ = −11 mV), and both maximum loading capacity (LC ∼ 17%) and entrapment efficiency (EE ∼ 100%). Kinetic release profiles show a slower release of Sor from nanoassemblies with respect to the free drug. SC6OH/Sor nanoassemblies have very low hemolytic activity and high efficiency in vitro in decreasing cell growth and viability of HCC cell lines, such as HepG2, Hep3B, and PLC/PRF/5, opening promising chances to their in vivo applications.
Nanoassemblies based on supramolecular complexes of non ionic amphiphilic cyclodextrins have here proposed to sustain the delivery of Sorafenib (Sor) in Hepatocellular Carcinoma (HCC). HCC is the third most common cause of cancer-related deaths worldwide. Advanced HCC is an extremely aggressive tumor with reduced prognosis and a very low response to ordinary therapies. Remedial chances had been rather scarce until the approval by the U.S. Food and Drug Administration (FDA) of Sorafenib (Nexavar, BAY43−9006) for the treatment of patients with advanced HCC. Unfortunately, various undesirable effects require to investigate other new therapeutic options for HCC. One of the drawback associated with the use of Sor is its low water-solubility (≅25 ng/mL for Sor free base and ≅ 65 ng/mL for Sor tosylate) and thereby extremely low bioavailability, which strongly decreases its therapeutic efficacy. Nowadays, two formulations are orally administrated but they possess low bioavailability due to the first-pass effect and cause a large adverse reaction in gastrointestinal irritation.
Taking into account the low water solubility of Sor and the high required dosage, it is a challenging task to increase its bioavailability by entrapment in suitable carriers, with the aim to control release and target Sor to liver tissues both at the early and at the end stage of disease. In this regard, the use of natural or modified cyclodextrins as host macrocycle able to accommodate and preserve the distinctive biological properties of anticancer guests is a well-established approach.
Cyclodextrins (CD), cyclic sugars constituted by seven glucose units, and their derivatives have been widely used to complex and deliver different kind of pharmaceutics, including anticancer drugs. In particular aCD substituted with hydrophobic (thioalkyl) and hydrophilic (oligoethylen glycol) portions at the primary and secondary rims of the macrocycle, respectively, can form different nanoarchitectures (such as micelles, micellar clusters, vesicles) in aqueous solution, depending on both the hydrophobic−hydrophilic balance and the charge of the CD carrier. In vitro, these drug delivery systems are able to entrap and deliver conventional anticancer drugs, phototherapeutics, antivirals, metals, and they have been proposed as potential non-viral agents for gene therapy. Covalent modifications with groups which recognizes protein overexpressed in cancer cells or luminescent labels confer a high potential for targeted drug delivery and drug monitoring, respectively. Generally, aCD nanocarriers (i.e. SC6OH used in this study) have sizes compatible with intravenous injection and have been proposed for optimizing anticancer drug distribution in the body through their potential to extravasate at the level of the tumor defective capillary bed and to deliver the drug to the site of action (EPR effect) (1-3).
Design and properties: In this study, we aimed to design nanoassemblies based on the aCD/Sor complexes to powerfully deliver Sor in aqueous media to HCC cells (Figure 1). The complex was characterized by UV−vis, fluorescence and NMR spectroscopy. Complexation of the anticancer drug in aCD was mainly evidenced by UV-Vis studies, as the band of free Sor centered at 264nm in organic solvent was red-shifted at 269 nm in the aqueous dispersion of Sor/aCD complex (Figure 1A). Additionally, this band showed hypochromicity with respect to free Sor, as evidenced by decreasing of extinction coefficient (ɛ), (ɛ of Sor was @ 41000, at λ=264 nm, with respect to ɛ of Sor/aCD complex that was @ 38000 at λ=269 nm). Scanning transmission electron microscopy (STEM) images confirm the presence of nanoaggregates or isolated nanospheres with an average size of ∼200 nm (Figure 1B), in agreement with dynamic light scattering results.
Figure 1. Sketched depiction of nanoassemblies design in aqueous solution from an amphiphilic cyclodextrin (SC6OH) and Sorafenib (Sor). A) UV−vis spectra showing the formation of complex: free Sor (trace a) in dichloromethane/acetonitrile and SC6OH/Sor complex (trace b) in aqueous solution (cell path, d = 0.2 cm; [Sor] = 200 μM, [SC6OH] = 200 μM, r.t. ≅ 25 °C, pH ≅ 6.7). B) Typical STEM micrographs of SC6OH/Sor nanoassemblies with a typical isolated nanosphere. C) Free Sor diffusion profile across the dialysis membrane and release profile of Sor from SC6OH/Sor in PBS at pH 7.4. (see Material and Methods in PMID: 26528591).
Sustained release and anti-hemolytic activity: Sor release profile from the aCD nanocarriers was investigated in physiological medium (PBS, pH 7.4). As shown in Figure 1C, no initial burst release was observed, and after 14 days, the whole release of Sor was about 38%. This result is very notable since faster release of Sor (which on the other hand has been experienced for free Sor) could result in quicker elimination of the drug in vivo, leading to a lower antitumor efficacy, and a worsening of patient compliance in terms of increasing number of administrations and consequent side effects. The effect of SC6OH/Sor nanoassemblies on human erythrocytes was investigated by comparison with that of empty SC6OH, to assess the hemocompatibility as a crucial prerequisite for an intravenously administered nanosystem. Empty SC6OH and Sor/SC6OH nanoassemblies showed no important hemolytic effects, indicating no interaction with red blood cell membranes.
Tumoral Cell Survival: To assess the in vitro cytotoxicity of the free drug, empty SC6OH and SC6OH/Sor nanoassemblies, short-term and long-term assays were carried out on three HCC cell lines, such as HepG2, Hep3B and PLC/PRF/5. Survival of all HCC cells ( see as an example Figure 2 A) decreases in a dose-dependent manner in the presence of either free Sor or SC6OH/Sor. These latter maintain an antitumor activity very close to the free drug (as an example, in PLC/PRF/5 cell lines IC50(Sor)@ 6.5 and IC50(aCD/Sor) @ 8). On the contrary, no cytotoxicity of the empty SC6OH was observed after 48 h, even at the highest tested concentration (Figure 2 B). Finally, a colony forming assay, which mimics the clonogenic survival of tumor cells in a solid tumor environment, was performed (Figure 2C), pointing out the declining of clonal growth and as a result the reduction of survival of HCC cells.
Figure 2: Effects of Sor, SC6OH, and SC6OH/Sor A) on the viability of PLC/PRF/5 cells(MTS assay) and (B,C) on the ability of human HCC cell line PLC/PRF/5 to form colony. Cells were plated overnight and exposed to the indicated concentration of solvent (DMSO), Sor, SC6OH, and SC6OH/Sor nanoassemblies for 48 h followed by growth in fresh culture media for 14 days. Surviving colonies were stained (B) and counted (C) (see Material and Methods in PMID: 26528591).
Conclusions: In summary, we designed and developed nanoassemblies composed by a cyclodextrin carrier and Sorafenib, a potent anticancer drug commonly utilized against hepatocarcinoma and other types of solid tumors. Host−guest complex was formed in aqueous solution by exploiting the role of the cyclodextrin cavity in including the hydrophobic drug. These novel nanoconstructs were highly stable in aqueous medium and sustained the release of Sor up to 2 weeks. Interestingly, these nanosystems show very low hemolytic activity and a high efficiency to inhibit the growth of three different HCC cell lines, similarly to free Sor. Considering that solid tumors present much more favorable conditions, due to the EPR effect, we believe, in perspective, that the preferential in vivo accumulation of cyclodextrin/Sorafenib nanoassemblies could result in a superior therapeutic efficacy than the free drug.
(1) Quaglia F, Ostacolo L, Mazzaglia A, Villari V, Zaccaria D, Sciortino MT 2009 The intracellular effects of non-ionic amphiphilic cyclodextrin nanoparticles in the delivery of anticancer drugs. Biomaterials, 30: 374-382
(2) Conte C, Ungaro F, Mazzaglia A, Quaglia F 2014 Photodynamic Therapy for Cancer: Principles, Clinical Applications and Nanotechnological Approaches. In Alonso MJ and Garcia-Fuentes M. eds. Nano-Oncologicals: New Targeting and Delivery Approaches, Advances in Delivery Science and Technology. NEW YORK, USA. CRS Springer pp 123-160 and ref therein
(3) Mazzaglia A, Bondì ML, Scala A, Zito F., Barbieri G, Crea F, Vianelli G, Mineo P, Fiore T, Pellerito C, Pellerito L., Costa MA 2013 Supramolecular Assemblies Based on Complexes of Nonionic Amphiphilic Cyclodextrins and a meso-Tetra(4- sulfonatophenyl)porphine Tributyltin(IV) Derivative: Potential Nanotherapeutics against Melanoma. Biomacromolecules, 14: 3820-3829
This work was supported by “Ministero dell’Istruzione, dell’Universitàe della Ricerca (Ministry for Education, Universities and Research) – MIUR FIRB-MERIT No. RBNE08YYBM to M.C., M.L.B., and A.M.
Dr Antonino Mazzaglia
CNR- ISMN at Dept of Chemical, Biological, Pharmaceutical
and Environmental Science (University of Messina),
Viale F. Stagno D’Alcontres 31, 98166, Messina, Italy