Nanoassemblies Based on Supramolecular Complexes of Nonionic

Nov 3, 2015 - CNR-ISMN Istituto per lo Studio dei Materiali Nanostrutturati - U.O.S. di ... capacity (LC ∼ 17%) and entrapment efficiency (EE ∼ 10...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/Biomac

Nanoassemblies Based on Supramolecular Complexes of Nonionic Amphiphilic Cyclodextrin and Sorafenib as Effective Weapons to Kill Human HCC Cells Maria Luisa Bondì,† Angela Scala,‡ Giuseppe Sortino,§ Erika Amore,∥ Chiara Botto,∥ Antonina Azzolina,⊥ Daniele Balasus,⊥ Melchiorre Cervello,⊥ and Antonino Mazzaglia*,§ †

CNR-ISMN Istituto per lo Studio dei Materiali Nanostrutturati - U.O.S. di Palermo, Via Ugo La Malfa 153, 90146 Palermo, Italy Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università di Messina, V.le F. Stagno D’Alcontres 31, 98166 Messina, Italy § CNR-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 ∥ Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy ⊥ Istituto di Biomedicina e Immunologia Molecolare “A. Monroy” - Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146 Palermo, Italy ‡

S Supporting Information *

ABSTRACT: 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.

■

INTRODUCTION Hepatocellular carcinoma (HCC) is the fifth most frequent cancer and the third most common cause of cancer-related deaths worldwide.1,2 HCC resulted in 696000 deaths worldwide in 2008. Advanced HCC is an extremely aggressive tumor with poor prognosis and a low or no response to common therapies. Therapeutic opportunity had been quite inadequate 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.3,4 Sorafenib (Sor) is an oral multikinase inhibitor which targets Raf kinases, as well as vascular endothelial growth factor (VEGFR)-2/-3, platelet-derived growth factor receptor β (PGFRβ), Fms-like tyrosine kinase 3 (Flt-3) and c-Kit, and currently is the only approved systemic treatment for HCC.5,6 Furthermore, Sor is an effective chemotherapeutic agent against © 2015 American Chemical Society

other types of tumors inhibiting proliferation, angiogenesis, and invasion of cancer cells.7,8 However, the benefit of Sor in the clinical practice is limited and various adverse effects associated with the monotherapy indicate the need to investigate other new therapeutic and/or modality options for HCC, including approaches inspired by combination treatment of cancer.9 One of the problems 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 significantly reduces its therapeutic efficacy.10 Nowadays, two formulations are orally administrated and absorbed Received: August 12, 2015 Revised: October 20, 2015 Published: November 3, 2015 3784

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791

Article

Biomacromolecules

dispersions, measured by pH-Meter-Metrohm 744, was 6.67 (±0.01). SC6OH/Sor film has been prepared by evaporation of a mixed organic solution of SC6OH in DCM and Sor in DCM/ACN (10:1 v/v), respectively. Spectroscopy. All the UV−vis have been carried out in quarz cuvettes (Hellma) with optical path of 0.2 cm, using a HewlettPackard mod. 8453 diode array spectrophotometer. Extinction coefficient of Sor, εSor(/DCM/ACN), and SC6OH/Sor, εSC6OH/Sor(H2O) have been determined by Lambert and Beer law in the range 6−200 μM. UV−vis spectra on SC6OH/Sor/Ada-COOH dispersion have been carried out by adding an excess of Ada-COOH (∼20 equiv) to SC6OH/Sor dispersion (200 μM). The samples have been kept under gentle stirring and analyzed at t = 0 min, 2 days, and 7 days. In parallel, SC6OH/Sor dispersions (without Ada-COOH) have been kept in the same conditions and analyzed at the same time range. To an aqueous dispersion of SC6OH/Sor nanoassemblies, Ada-COOH was added and left under stirring over 7 days, followed by equilibration at r.t. The precipitate was isolated after 1 month of settling and analyzed by UV− vis in DCM/ACN (90/10, v/v). UV−vis spectrum of aqueous supernatant was also carried out for comparison. Fluorescence emission spectra and resonance light scattering measurements (RLS) have been registered on a spectrofluorimeter JASCO mod. FP-750, by using a 1 cm path length quartz cell. NMR Studies. NMR spectra were recorded on a Varian 500 MHz spectrometer at room temperature (r.t. ≅ 25 °C). The chemical shifts (δ) and the coupling constants are expressed in ppm and Hertz, respectively. The spectra were recorded on the following samples: [Sor] = 1 mM (CDCl3, V = 750 μL) and [SC6OH/Sor] = 1 mM (D2O, V = 750 μL). ROESYAD (Adiabatic Rotating frame nuclear Overhauser effect spectroscopy) experiment was registered by overnight acquisition. Sor 1H NMR (CDCl3; 500 MHz): δ 8.98 (s br, 1H, NHCH3 H-k), 8.49 (d, 1H, J = 6.2, H-h), 8.32 (d, 2H, J = 8.8, H-f), 8.09 (s, 1H, NH H-d), 7.85 (m, 2H, H-i, H-j), 7.66 (d, 1H, J = 8.9, H-b), 7.52 (s, 1H, NH H-e), 7.46 (d, 2H, J = 8.8, H-g), 7.13 (m, 1H, H-c), 7.04 (m, 1H, H-a), 3.02 (d, 3H, J = 4.4, CH3NH). SC6OH/Sor 1H NMR (D2O; 500 MHz): only shifted peaks δ 9.53 (H-h), 8.50 (H-f), 8.12 (H-i, H-j), 7.55 (H-b), 7.23 (H-g), 7.14 (H-c), 6.95 (H-a). Particle Size Analysis. The mean diameter and width of distribution (polydispersity index, PDI) of the empty SC6OH and SC6OH/Sor nanoassemblies were measured by Photon Correlation Spectroscopy (PCS) by a Zetasizer Nano ZS (Malvern Instrument, Malvern, U.K.) utilizing a Non-Invasive Back-Scattering (NIBS) technique. The measurements were performed at a 173° angle with respect to the incident beam at 25 ± 1 °C for each dispersion of SC6OH and SC6OH/Sor nanoassemblies using bidistilled water or a 0.9 wt % NaCl or a PBS at pH 7.4 aqueous solutions as the dispersing media. Each dispersion was kept in a cuvette and analyzed in triplicate. The deconvolution of the measured correlation curve to an intensity size distribution was achieved by using a non negative least-squares algorithm. ζ-Potential Measurements. The ζ-potential values have been determined by utilize a laser Doppler velocimetry and phase analysis light scattering (M3-PALS technique). At this purpose, a Zetasizer Nano ZS Malvern Instrument equipped with a He−Ne laser at a power P = 4.0 mW and with λ = 633 nm was used. Empty SC6OH and SC6OH/Sor nanoassemblies were dispersed in bidistilled water, or in 0.9 wt % NaCl, or in PBS at pH 7.4 aqueous solutions with the conductivity adjusted to 50 mS/cm. The results are reported as the mean of three separate measurements on three different batches ± the standard deviation (SD). Microscopy. STEM analyses were carried out with the Zeiss-Gemini 2 electron microscope, operating at 30 kV and at a working distance of 4 mm. SC6OH/Sor nanoassemblies were dispersed in H2O ([SC6OH/Sor] = 20 μM) by ultrasonic bath, deposited on a 400 mesh holey-carbon grid and left to dry overnight at r.t. Drug Loading and Encapsulation Efficiency. An appropriate HPLC method was used to detect Sor and to study both its stability,

through the gastrointestinal tract, but they can cause a great undesirable reaction in that area because of their low bioavailability.11 These problems might be overcome by use of nanoparticles (NPs) to control release10,12 and target Sor to liver tissues both at the early and at the end stage of disease.13,14 Sor-loaded NPs,15,16 polymeric micelles,17 or lipid nanocarriers18−20 represent some valuable choices to optimize bioavailability, pharmacokinetics, and targeting drug to malignant cells or metastasis.21 Cyclodextrins (CD), cyclic oligosaccharides constituted by seven D-glucose units linked by α-1,4 glucosidic bonds, and their conjugates have been widely used to complex and deliver different kind of pharmaceutics,22,23 including anticancer drugs.24,25 Despite of a few studies demonstrate the utilize of CD as pharmaceutical excipients in formulation with Sor,26 only rare reports show the ability of natural or commercial CD to form complexes with anticancer drugs.27 Over the past two decades, scientists developed a plethora of amphiphilic CD (aCD) able to increase bioavailability and modulate pharmacokinetics of chemotherapeutics.28 Recently, some of us investigated nanoassemblies based on nonionic aCD that efficiently deliver antimitotic,29 antivirals,30 and porphyrinoids drugs,31,32 the latters by using both photodependent33 or -independent34 therapeutic approaches. Usually, nonionic aCD substituted with short thioalkyl chains show a lower darktoxicity with respect to the cationic analogues,35 thus they could be ideal to deliver drugs (i.e., Sor) which result toxic at high doses (IC50 of Sor in HCC cells, after predissolution in DMSO, is between ∼5 and 9 μM, see Table S1). In this paper, we newly aim to entrap Sor in a nonionic aCD with maxima efficiency, releasing it in physiological media to finally act against HCC cells. Nanoassemblies based on SC6OH/Sor have been easily prepared by hydration of a thin organic film in aqueous media followed by ultrasonication, and characterized to elucidate size and surface charge. Spectroscopic evidence (UV−vis, steadystate fluorescence emission and related static anisotropy, circular dichroism, and NMR) agree with the formation of a host−guest complex between aCD and Sor, plausibly by exploiting the interaction of the drug with the macrocyclic cavity. Finally, in vitro biological characterization was carried out. In particular, to evaluate the toxicity and biological activity of the empty and loaded systems, hemolytic activity, cell viability, and clonogenic assays were carried out using human HCC cells.

■

MATERIALS AND METHODS

General Remarks. All solvents were purified and dried using standard techniques. All solutions used for spectroscopic characterizations were prepared in pure microfiltered water (Galenica Senese) and analyzed at 298 K. The heptakis(2-O-oligo(ethylene oxide)-6hexylthio)-β-CD (SC6OH, MWn=2 = 2763 amu) was synthesized according to the general procedures.36 Sorafenib-free base (Sor) was purchased from LC Laboratories, Division of PKC Pharmaceuticals, Inc., Woburn, MA (U.S.A.). 1-Adamantane carboxylic acid (AdaCOOH), CDCl3, and D2O for NMR measurements were acquired from Sigma-Aldrich. Acetonitrile (ACN), dichloromethane (DCM), and ethanol (EtOH) for HPLC were purchased from Merck (Milan, Italy). Nanoassemblies Preparation and Characterization. Nanoassemblies based on SC6OH/Sor were prepared at SC6OH/Sor 1:1 molar ratio ([Sor] = [SC6OH] = 200 μM) according to the reported procedures used to entrap other drugs.34 Briefly, SC6OH/Sor complex was prepared by the dispersion of a thin organic film in ultrapure water at room temperature followed by 10 min of sonication in ultrasonic bath and used as freshly obtained or lyophilized. pH of the aqueous 3785

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791

Article

Biomacromolecules Scheme 1. Sketched View of Nanoassemblies Formation in Aqueous Solution from SC6OH and Sor

loading capacity (LC%), as well as drug release profiles from SC6OH/ Sor nanoassemblies. HPLC analysis was carried out at r.t. using a HPLC (UFLC-Prominence system, Shimadzu Instrument, Kyoto, Japan) equipped with two pumps LC-20 AD, an UV−visible detector SPD-20 AV, an autosample SIL-20A HT and a column Gemini C18 (μBondpack 5 μm, 250 × 4.60 mm i.d., Phenomenex) as stationary phase. A mixture of ACN/H2O 95:5 with a flow rate of 1.2 mL/min was selected as the mobile phase. The peak was measured at a wavelength of 264 nm and quantitatively determined by calibration curve of free Sor in ACN/DCM/EtOH 4:4:2 (v/v/v; tr = 7 min). The linearity of the method was studied in the range 10−200 μg/mL. LC% was measured by dissolving a film of SC6OH/Sor nanoassemblies in 200 μL of DCM/ACN/EtOH (4:4:2 v/v), with [Sor] = 200 μM. Hence, the solution was sonicated for 5 min, filtered with 0.45 μm PTFE filters and analyzed by the above-described HPLC method. To verify the possible interference of aCD, empty SC6OH was dissolved in the same organic mixture and injected to HPLC. The results are reported as actual loading percentage (L.C.%, mg of drug encapsulated per 100 mg of nanoassemblies) and encapsulation efficiency (EE%, the ratio of actual to theoretical loading). To confirm that the drug is not absorbed within the PTFE filters, organic solutions of Sor at known concentrations were filtered and analyzed by HPLC. Sor Release Kinetics. Briefly, SC6OH/Sor nanoassemblies ([Sor] = [SC6OH] = 40 μM) dispersed in PBS (1 mL) was put into a dialysis tube (MWCO 3 kDa) and immersed into 20 mL of preheated PBS (pH = 7.4) at 37 ± 0.1 °C under continuous stirring in a Benchtop incubator Orbital Shaker (model 420). At fixed times, 1 mL of release medium was withdrawn and replaced with an equal volume of fresh PBS and analyzed by UV−vis. The release behavior of free Sor was also studied by a control experiment in the same conditions. Biological Studies. Hemolytic Test. Hemolytic test has been carried out as previously described.20 Cell Culture. The human HCC cell lines HepG2, Hep3B and PLC/ PRF/5 were maintained in Roswell Park Memorial Institute (RPMI) medium (Sigma, Milan, Italy) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, Milan, Italy), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin (all reagents were from Sigma) in a humidified atmosphere at 37 °C in 5% CO2. Cells having a narrow range of passage number were used for all experiments. Cell Viability Assays. Cells (5 × 103/well) were seeded into each well of 96-well microtiter plates and then incubated overnight. At time 0, the medium was replaced with fresh complete medium, and various concentrations of free Sor (12.5, 25, 50, 100, and 200 μM, in DMSO) and SC6OH/Sor nanoassemblies were diluted with one volume of 2× concentrated RPMI complete medium and added to the cells. Empty SC6OH were also evaluated on HCC cell lines growth at the same concentrations. Lyophilized samples were resuspended in sterile

conditions sonicated for 20 min, diluted in sterile culture medium and added to the cells. Cells were cultured for 48 h. At the end of treatment, MTS assays were performed as previously described.20 Bacterial growth was not observed in any sample. Colony Formation Assays. The inhibitory effect of SC6OH/Sor nanoassemblies on cell growth was also assessed using a clonogenic assay. PLC/PRF/5 cells (500/well) were plated onto six-well plates in growth medium and after overnight attachment cells were exposed to various concentrations of solvent (DMSO), Sor, empty SC6OH and SC6OH/Sor nanoassemblies for 48 h. The cells were then washed with medium and allowed to grow for 14 days. Afterward, colonies were stained and counted as previously described.20 All the experiments were performed in duplicate and repeated twice.

■

RESULTS AND DISCUSSION aCD/Sor Supramolecular Complexes: Design and Spectroscopic Investigations. Taking into account the low

Figure 1. UV−vis spectra of free Sor (trace a) in DCM/ACN (90/10, v/v) 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). In the inset, determination of extinction coefficients of free Sor in DCM/ACN (a) and SC6OH/Sor complex in aqueous solution (b) by Lambert−Beer is reported, respectively: εSor(λ=264 nm) = 41280 ± 480 and εcomplex(λ=269 nm) = 37500 ± 800.

water solubility of Sor and the high required dosage against HCC cells, it is a challenging task to increase its bioavalaibility by entrapment in suitable carriers. The use of natural or modified cyclodextrins as host macrocycle able to accommodate and preserve the distinctive biological properties of 3786

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791

Article

Biomacromolecules

complexation of Sor with aCD has been further confirmed by higher values of steady-state anisotropy for SC6OH/Sor than for the free drug (inset of Figure 2). To get insight on the complexation sites of Sor in SC6OH, we registered UV−vis spectra of SC6OH/Sor complex in the presence of Ada-COOH, a competitive guest for the macrocyclic cavity. Figure 3 shows UV−vis spectra of SC6OH/Sor complex before and after addition of Ada-COOH. It is wellestablished that adamantyl moiety of Ada-COOH is able to be accommodated in the basket of amphiphilic CD, forming stable inclusion complexes.33,41 As a result, the freshly added AdaCOOH slightly increases the extinction of SC6OH/Sor (Figure 3, trace c), plausibly due to a rearrangement of Sor within SC6OH assemblies. After 2 days, Sor diffuses out and the incipient precipitation leads to a strong extinction decrease (Figure 3, trace d) until to the disappearance of the peculiar Bband of Sor and to a strong turbidity increase (after 7 days, Figure 3, trace e). Furthermore, RLS method permits to detect small scattering light increments in the analyzed colloidal dispersion, due to fluorophores rearrangement, by synchronously fluorescence measurement in a wavelength range.42 Figure S2 shows RLS spectrum of SC6OH/Sor, registered at t = 0 min, after addition of Ada-COOH. RLS evidence a light scattering increase (i.e., at 400 nm) due probably to the release of Sor induced by competition with Ada-COOH for the aCD cavity. These evidence might be ascribed to the saturation of CD binding sites by Ada-COOH, this process being in equilibrium with the displacement of the drug from aCD cavity and its following precipitation. These observations were confirmed (i) by the visual inspection of the dispersions after treatment with adamantyl-derivative (see Figure S1); (ii) by analyzing both the supernatant and the precipitate obtained by settling of the SC6OH/Sor/Ada-COOH system. In particular, the precipitate was redissolved in DCM/ACN and analyzed by UV−vis, recovering ∼75% of total entrapped Sor (see Figure S3). On the other hand, SC6OH/Sor complex (without addition of Ada-COOH) is stable in ultrapure water for about 2 weeks (data not shown). Furthermore, the interaction of Sor with SC6OH was elucidated by NMR spectroscopy. 1H NMR spectrum of SC6OH/Sor in D2O (Figure 4A) was compared with that of free SC6OH in the same deuterated solvent and the chemical shifts of CD protons remain mostly unshifted, even if they are fairly broad because of aggregates formation. As free Sor is insoluble in D2O, the same comparison was not possible, but interestingly, the presence of signals in the region 9.6−6.8 ppm unambiguously assigned to Sor protons in the 1H NMR, spectrum of the complex in D2O (see magnification, Figure 4B), clearly indicated the interaction of the drug with the carrier. Furthermore, in the complex, the peaks multiplicity is lost for the drug and a marked broadening of all signals are observed, as a result of complexation phenomena. The 1H NMR spectrum of Sor in CDCl3 with the related assignments was also reported (Figure 4C) for comparison. Obviously the chemical shifts slightly change depending on the used solvent. In particular, the α proton of the pyridine ring (H-h) experiences an evident deshielding (∼1 ppm in D2O vs CDCl3), likely because the protonation of the pyridine ring in D2O leads to a well-known downfield shift of their proton signals.43,44 ROESY spectrum (Figure 4D) showed valuable correlations between H-i, H-j and H-f of Sor with H-3, H-5 of the aCD

Figure 2. Fluorescence emission spectra and correspondent steadystate anisotropy (in the inset) of (a) free Sor (λexc = 295 nm) in DCM/ACN (90/10, v/v) and (b) SC6OH/Sor complex (λexc = 303 nm) in aqueous dispersion ([Sor] = 200 μM, [SC6OH] = 200 μM, r.t. ≅ 25 °C).

Figure 3. UV−vis spectra of SC6OH/Sor (trace b) in aqueous solution and after addition of Ada-COOH (trace c, freshly added; trace d, stirred for 2 days; trace e: stirred for 7 days; cell path, d = 0.2 cm; [Sor] = 200 μM, [SC6OH] = 200 μM, r.t.).

anticancer guests is a well-established approach.37−39 With this in mind, in this study, we aim to design nanoassemblies based on aCD/Sor complex to efficiently deliver Sor in aqueous media into HCC cells (Scheme 1). First, we investigated the interaction of Sor with SC6OH in aqueous solution by UV−vis, steady state fluorescence and NMR spectroscopy. UV−vis spectra of free Sor in DCM/ACN and SC6OH/Sor in aqueous dispersion are reported in Figure 1. Free Sor, dissolved in organic solvent mixture, shows a Bband centered at 264 nm, belonging to an aromatic moiety of the drug,40 red-shifted at 269 nm upon complexation with SC6OH. Additionally, this band shows hypochromicity, after interaction with amphiphile with respect to free Sor, as evidenced by decreasing of extinction coefficient. Fluorescence emission spectrum of free Sor shows one band centered at 487 nm with a shoulder at ∼433 nm (Figure 2). These two bands result remarkably blu-shifted at ∼470 and ∼418 nm respectively, by interaction with SC6OH. Furthermore, SC6OH/Sor complex shows an increase of the emission intensity bands, ascribed to the environment change after entrapment in aCD. It is also noteworthy that the positions of the two emission bands were not dependent on the excitation energy (data not shown), suggesting the existence of a single population of emitting drug into the nanoassemblies. The 3787

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791

Article

Biomacromolecules

Figure 4. (A) Full 1H NMR spectrum of SC6OH/Sor in D2O; (B) related magnification of the aromatic region (9.6−6.8 ppm); (C) magnification of the aromatic region (9.6−6.8 ppm) of the 1H NMR spectrum of Sor in CDCl3 and chemical structure of Sor with protons assignment; (D) ROESYAD spectrum of SC6OH/Sor in D2O.

Table 1. Overall Properties of Empty SC6OH and SC6OH/ Sor Nanoassemblies: Mean Diameter, Polydispersity Index (PDI), Zeta Potential in Bidistilled Water, in Aqueous Solution of NaCl 0.9 wt %, and in PBS pH 7.4a sample SC6OH

SC6OH/Sor

dispersing medium

mean diameter (nm)

PDI

zeta potential (mV; ±S.D)

PBS pH 7.4 NaCl 0.9% H2O PBS pH 7.4 NaCl 0.9% H2O

224 223 204 240 250 222

0.28 0.23 0.27 0.29 0.28 0.21

−1.1 ± 1.4 −1.3 ± 1.9 −6.2 ± 1.0 −3.4 ± 0.9 −4.3 ± 1.8 −16.2 ± 2.2

Figure 5. STEM analysis of SC6OH/Sor nanoassemblies (A) with a typical isolated nanosphere (B).

a

SC6OH/Sor LC% (expressed as weight percent ratio between entrapped Sor and the total dried sample weight) was ≅17% and the EE% (expressed as weight percent ratio between entrapped Sor and total amount of Sor used to prepare SC6OH/Sor nanoassemblies) was ≅100%.

potential) are reported in Table 1. Empty SC6OH exhibited a size distribution with a mean diameter of approximately 200 nm in water, that slightly increased in drug-loaded SC6OH (Figure S4). In addition, the ζ-potential values were close to zero in saline media and decreased in bidistilled water. STEM images confirm the presence of aggregates or isolated nanospheres with an average size of ∼200 nm (Figure 5), in agreement with DLS data (Figure S4). Sor release profile from the nanoassemblies was studied in physiological medium (PBS, pH 7.4), under sink conditions, using the dialysis bag technique, as reported in literature for other Sor-loaded nanosystems.14 At fixed times, 1 mL of release

cavity, pointing out the inclusion of the drug into CD. However, other sites of interactions of Sor with aCD side chains cannot be excluded. Nanoassemblies of SC6OH/Sor: Properties and Release Studies. SC6OH/Sor nanoassemblies have been prepared by hydration of a thin organic film of SC16OH/Sor and characterized to elucidate loading capacity, size and surface charge. The properties of empty SC6OH and SC6OH/Sor nanoassemblies (mean diameter, polydispersity index, and ζ3788

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791

Article

Biomacromolecules

Figure 6. Free Sor diffusion profile across the dialysis membrane and release profile of Sor from SC6OH/Sor in PBS at pH 7.4.

Figure 7. Hemolysis percentage after incubation with red blood cells for 1 h at 37 °C with free Sor, empty SC6OH, SC6OH/Sor nanoassemblies (drug concentrations: 5, 10, and 20 μM). Data are represented as mean ± SD for three separate experiments.

medium was withdrawn, replaced with an equal volume of fresh PBS, and analyzed by UV−vis. The amount of released Sor was expressed as percentage ratio between the weight of released drug at prefixed times and the total amount of entrapped Sor (Figure 6). As shown, no initial burst release was observed, and after 14 days, the cumulative release of Sor was about 38%. This result is very remarkable since faster release of Sor (which on the other hand has been experienced for free Sor) could result in faster elimination of the drug in vivo, leading to a lower antitumor efficacy, and a worsening of patient compliance in terms of frequency of administrations and subsequent side effects. In Vitro Biological Studies. Hemolysis Studies. The effect of SC6OH/Sor system on human erythrocytes was investigated by comparison with that of empty SC6OH, to assess the hemocompatibility of the nanoassemblies as a crucial prerequisite for an intravenously administered nanosystem. The incubation of SC6OH and SC6OH/Sor was performed under appropriate settings quantifying the hemoglobin release.16 Empty SC6OH and drug-loaded SC6OH showed no significant hemolytic effects (Figure 7), indicating no interaction with red blood cell membranes. The percentage of hemolysis was always less than 2.0%, even at the highest tested concentration (0.47 and 1.38% for SC6OH and SC6OH/Sor, respectively). Moreover, no erythrocyte aggregation was detected after incubation with empty or drug-loaded aCD (see Figure S6, for visual inspection of hemolysis assay).

Figure 8. Effects of Sor, SC6OH, and SC6OH/Sor on the viability of HepG2 (A), Hep3B (B), and PLC/PRF/5 (C) cells. Cells were grown and treated for 48 h with the indicated concentration of free Sor, SC6OH and SC6OH/Sor. Cell viability was expressed as a percentage of the absorbance measured in the control cells. Values were expressed as means ± SD of three separate experiments, each performed in triplicate.

Viability Assays. To evaluate the in vitro cytotoxicity of the free drug, empty SC6OH and SC6OH/Sor nanoassemblies, short-term and long-term assays studies were performed on three HCC cell lines, such as HepG2, Hep3B and PLC/PRF/5. For short-term analysis, cell vitality was evaluated by MTS assays after treatment for 48 h, whereas for long-term analysis, clonogenic assays were used to measure the colony formation after 14 days of treatment. In this regard, clonogenic assay is considered the “gold standard” test to assess the drug ability to kill cancer cells in vitro, by measuring the degree of clonal growth and thus the replicative capacity and survival of tumor 3789

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791

Article

Biomacromolecules

up to 2 weeks. Interestingly, these systems 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 SC6OH/Sor nanoassemblies could result in a superior therapeutic efficacy than the free drug.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01082. Visual inspection of SC6OH/Sor dispersions before and after addition of Ada-COOH (Figure S1); Resonance Light Scattering spectra of SC6OH/Sor before and after addition of Ada-COOH (Figure S2); UV−vis spectra of precipitate, dissolved in DCM/ACN (90/10, v/v), and supernatant (Figure S3); Size distribution from DLS of SC6OH/Sor nanoassemblies (Figure S4); Visual inspection of hemolysis assay (Figure S5); IC50 average values for free Sor and SC6OH/Sor nanoassemblies (Table S1) (PDF).

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +39 0903974108. Fax: +39 0903974108. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Dr Enza Fazio (Università di Messina) for STEM analysis. This work was supported in part by grants from the Italian “Ministero dell’Istruzione, dell’Università e della Ricerca (Ministry for Education, Universities and Research) − MIUR FIRB-MERIT No. RBNE08YYBM to M.C., M.L.B., and A.M.

Figure 9. Effects of Sor, SC6OH, and SC6OH/Sor nanoassemblies 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, empty SC6OH, and SC6OH/Sor nanoassemblies for 48 h followed by growth in fresh culture media for 14 days (see Materials and Methods). Surviving colonies were stained (A) and counted (B). Data are expressed as a percentage of colony in untreated cells and are the mean ± SD of two determinations.

■

REFERENCES

(1) El-Serag, H. B. Gastroenterology 2012, 142, 1264−1273. (2) Cervello, M.; McCubrey, J. A.; Cusimano, A.; Lampiasi, N.; Azzolina, A.; Montalto, G. Oncotarget. 2012, 3, 236−260. (3) Llovet, J. M.; Ricci, S.; Mazzaferro, V.; Hilgard, P.; Gane, E.; Blanc, J. F.; de Oliveira, A. C.; Santoro, A.; Raoul, J. L.; Forner, A.; Schwartz, M.; Porta, C.; Zeuzem, S.; Bolondi, L.; Greten, T. F.; Galle, P. R.; Seitz, J. F.; Borbath, I.; Häussinger, D.; Giannaris, T.; Shan, M.; Moscovici, M.; Voliotis, D.; Bruix, J. N. Engl. J. Med. 2008, 359, 378− 390. (4) Cheng, A. L.; Kang, Y. K.; Chen, Z.; Tsao, C. J.; Qin, S.; Kim, J. S.; Luo, R.; Feng, J.; Ye, S.; Yang, T. S.; Xu, J.; Sun, Y.; Liang, H.; Liu, J.; Wang, J.; Tak, W. Y.; Pan, H.; Burock, K.; Zou, J.; Voliotis, D.; Guan, Z. Lancet Oncol. 2009, 10, 25−34. (5) Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.; Chen, C.; Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.; Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.; Voznesensky, A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. Cancer Res. 2004, 64, 7099− 7109. (6) Llovet, J. M.; Di Bisceglie, A. M.; Bruix, J.; Kramer, B. S.; Lencioni, R.; Zhu, A. X.; Sherman, M.; Schwartz, M.; Lotze, M.; Talwalkar, J.; Gores, G. J. J.Natl. Cancer Inst. 2008, 100, 698−711. (7) Kane, R. C.; Farrell, A. T.; Saber, H.; Tang, S.; Williams, G.; Jee, J. M.; Liang, C.; Booth, B.; Chidambaram, N.; Morse, D.; Sridhara, R.;

cells. As shown in Figure 8, survival of all HCC cells decreases in a dose-dependent manner in the presence of either free Sor or SC6OH/Sor nanoassemblies, the latter maintaining an antitumor activity very close to the free drug (see IC50, Table S1). Conversely, no cytotoxicity of the empty SC6OH was observed after 48 h, even at the highest concentration (Figure 8). Finally, a colony forming assay, which mimics the clonogenic survival of tumor cells in a solid tumor environment, was carried out (Figure 9), pointing out the decreasing of clonal growth and consequently the reduction of survival of HCC cells.

■

CONCLUSIONS A proper design and development of nanoassemblies based on amphiphilic cyclodextrin complexing sorafenib, a powerful anticancer drug commonly utilized against hepatocarcinoma and other types of solid tumors, were here proposed. Host− guest aCD/Sor complex was formed in aqueous solution by exploiting the cooperation of the CD cavity. These nanoassemblies are highly stable in aqueous medium, retaining Sor 3790

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791

Article

Biomacromolecules Garvey, P.; Justice, R.; Pazdur, R. Clin. Cancer Res. 2006, 12, 7271− 7278. (8) White, P. T.; Cohen, M. S. Expert Opin. Drug Discovery 2015, 10, 427−439. (9) Cervello, M.; Bachvarov, D.; Lampiasi, N.; Cusimano, A.; Azzolina, A.; McCubrey, J. A.; Montalto, G. PLoS One 2013, 8, e65569. (10) PCT (International application published under the patent cooperation treaty). International publication number WO 2008/ 008733 A2, 2008. (11) Liu, Y. O.; Fan, J. M.; Wang, X. Q.; Zhang, Q. J. Chin. Pharm. Sci. 2011, 20, 164−170. (12) Wang, X. Q.; Fan, J. M.; Liu, Y. O.; Zhao, B.; Jia, Z. R.; Zhang, Q. Int. J. Pharm. 2011, 419, 339−346. (13) Poelstra, K.; Prakash, J.; Beljaars, L. J. Controlled Release 2012, 161, 188−197. (14) Giannitrapani, L.; Soresi, M.; Bondì, M. L.; Montalto, G.; Cervello, M. World J. Gastroenterol. 2014, 20, 7242−725. (15) Mato, E.; Puras, G.; Bell, O.; Agirre, M.; Hernández, R. M.; Igartua, M.; Moreno, R.; Gonzalez, G.; de Leiva, A.; Pedraz, J. L. J. Nanomed. Nanotechnol. 2015, 6, 1000281. (16) Kim, D. H.; Kim, M. D.; Choi, C. W.; Chung, C. W.; Ha, S. H.; Kim, C. H.; Shim, Y. H.; Jeong, Y.; Kang, D. H. Nanoscale Res. Lett. 2012, 7, 91−96. (17) Craparo, E. F.; Sardo, C.; Serio, R.; Zizzo, M. G.; Bondì, M. L.; Giammona, G.; Cavallaro, G. Int. J. Pharm. 2014, 466, 172−180. (18) Zhang, H.; Zhang, F. M.; Yan, S. J. Int. J. Nanomed. 2012, 7, 2901−2910. (19) Bondì, L.; Azzolina, A.; Craparo, E. F.; Botto, C.; Giammona, G.; Cervello, M. J. Nanobiotechnology 2014, 12, 1−9. (20) Bondì, M. L.; Botto, C.; Amore, E.; Emma, M. R.; Augello, G.; Craparo, E. F.; Cervello, M. Int. J. Pharm. 2015, 493, 75−85. (21) Zhang, Z.; Niu, B.; Chen, J.; He, X.; Bao, X.; Zhu, J.; Yu, H.; Li, Y. Biomaterials 2014, 35, 4565−4572. (22) Loftsson, T.; Brewster, M. E. J. Pharm. Pharmacol. 2010, 62, 1607−1621. (23) Jansook, P.; Brewster, M. C. Loftsson, T. In Cyclodextrins in Pharmaceutics, Cosmetics, and Biomedicine Current and Future Industrial Applications; Bilensoy, E., Ed.; John Wiley and Sons: New York, 2011; pp 145−158. (24) Tan, H.; Qin, F.; Chen, D.; Han, S.; Lu, W.; Yao, X. Carbohydr. Polym. 2013, 93, 679−685. (25) Salmaso. S.; Sonvico, F. In Cyclodextrins in Pharmaceutics, Cosmetics, and Biomedicine Current and Future Industrial Applications; Bilensoy, E., Ed.; John Wiley and Sons: New York, 2011; pp 251−273 and ref. therein. (26) Kim, A.; McCully, C.; Cruz, R.; Cole, D. E.; Fox, E.; Balis, F. M.; Widemann, B. C. Invest. New Drugs 2012, 30, 524−528. (27) Hashemi, F.; Tamaddon, A. M.; Yousefi, G. H.; Farvadi, F. Proc. NAP 1, 02NNBM06; Nanomaterials: Application and Properties '2012, Crimea, Ukraine, Sept 17−22, 2012, SUMY State Univ.: Ukraine, 2012. (28) Bilensoy, E.; Hincal, A. A. Expert Opin. Drug Delivery 2009, 6, 1161−1173. (29) Quaglia, F.; Ostacolo, L.; Mazzaglia, A.; Villari, V.; Zaccaria, D.; Sciortino, M. T. Biomaterials 2009, 30, 374−382. (30) Scala, A.; Cordaro, M.; Mazzaglia, A.; Risitano, F.; Venuti, A.; Sciortino, M. T.; Grassi, G. MedChemComm 2011, 2, 172−175. (31) Mazzaglia, A. In Cyclodextrins in Pharmaceutics, Cosmetics, and Biomedicine Current and Future Industrial Applications; Bilensoy, E., Ed.; John Wiley and Sons: New York, 2011; pp 343−361. (32) Conte, C.; Ungaro, F.; Mazzaglia, A.; Quaglia, F. In NanoOncologicals: New Targeting and Delivery Approaches. Advances in Delivery Science and Technology; Alonso, M. J., Garcia-Fuentes, M., Eds; CRS Springer: New York, 2014; pp 123−160. (33) Kandoth, N.; Vittorino, E.; Sciortino, M. T.; Parisi, T.; Colao, I.; Mazzaglia, A.; Sortino, S. Chem. - Eur. J. 2012, 18, 1684−1690.

(34) Mazzaglia, A.; Bondì, M. L.; Scala, A.; Zito, F.; Barbieri, G.; Crea, F.; Vianelli, G.; Mineo, P.; Fiore, T.; Pellerito, C.; Pellerito, L.; Costa, M. A. Biomacromolecules 2013, 14, 3820−3829. (35) Conte, C.; Scala, A.; Siracusano, G.; Leone, N.; Patanè, S.; Ungaro, F.; Miro, A.; Sciortino, M. T.; Quaglia, F.; Mazzaglia, A. RSC Adv. 2014, 4, 43903−43911. (36) Mazzaglia, A.; Donohue, R.; Ravoo, B. J.; Darcy, R. Eur. J. Org. Chem. 2001, 2001, 1715−1721. (37) Mazzaferro, S.; Bouchemal, K.; Gallard, J.-F.; Iorga, B. I.; Cherona, M.; Gueutina, C.; Steinmesse, C.; Gilles Ponchel, G. Int. J. Pharm. 2011, 416, 171−180. (38) Kang, Y.; Zhang, X.-M.; Zhang, S.; Ding, L.-S.; Li, B.-J. Polym. Chem. 2015, 6, 2098−2107. (39) Kang, Y.; Ma, Y.; Zhang, S.; Ding, L.-S.; Li, B.-J. ACS Macro Lett. 2015, 4, 543−547. (40) Shi, J.-H.; Chen, J.; Wang, J.; Zhu, Y.-Y. Spectrochim. Acta, Part A 2015, 136, 443−450. (41) Roling, O.; Wendeln, C.; Kauscher, U.; Seelheim, P.; Galla, H.-J; Ravoo, B. J. Langmuir 2013, 29, 10174−10182. (42) Shi, J.-H.; Chen, J.; Wang, J.; Zhu, Y.-Y.; Wang, Q. Spectrochim. Acta, Part A 2015, 149, 630−637. (43) Gil, V. M. S.; Murrell, J. N. Trans. Faraday Soc. 1964, 60, 248− 255. (44) Hesse, M.; Meier, H.; Zeeh, B. Spectroscopic Methods in Organic Chemistry, 2nd ed.; Thieme: Stuttgart, Germany, 2007.

3791

DOI: 10.1021/acs.biomac.5b01082 Biomacromolecules 2015, 16, 3784−3791