Intercalation of bioactive molecules into nanosized ZnAl hydrotalcites

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Intercalation of bioactive molecules into nanosized ZnAl hydrotalcites for combined chemo and photo cancer treatment Cecilia Martini, Claudia Ferroni, Marzia Bruna Gariboldi, Anna Donnadio, Annalisa Aluigi, Giovanna Sotgiu, Fabiola Liscio, Paolo Dambruoso, Maria Luisa Navacchia, Tamara Posati, and Greta Varchi ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01601 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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ACS Applied Nano Materials

Intercalation of Bioactive Molecules into Nanosized ZnAl Hydrotalcites for Combined Chemo and Photo Cancer Treatment Cecilia Martini,‡,¶,† Claudia Ferroni,‡,¶ Marzia Bruna Gariboldi,# Anna Donnadio,§ Annalisa Aluigi,¶ Giovanna Sotgiu,¶ Fabiola Liscio,¬ Paolo Dambruoso,¶ Maria Luisa Navacchia,¶ Tamara Posati,*¶ and Greta Varchi*¶

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Institute of Organic Synthesis and Photoreactivity, National Research Council, Via Gobetti 101, 40129 Bologna, Italy.

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Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy.

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Department of Biotechnology and Life Science, University of Insubria, Via Ravasi 2, 21100 Varese, Italy.

ACS Paragon Plus Environment

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Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy.

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Institute for Microelectronics and Microsystems, National Research Council, Via Gobetti 101, 40129 Bologna, Italy.

KEYWORDS hydrotalcites (HTlc), norcantharidin (NCTD), tetra-sulfonated aluminum phthalocyanine (AlPcS4), synergistic effect, photodynamic therapy (PDT), cancer cells

ABSTRACT

Hydrotalcites-like compounds, also known as anionic clays or layered double hydroxides, represent the only example of lamellar solid with positively charged layers and exchangeable interlayer anions. In this study, a nanostructured HTlc with formula [Zn0.72Al0.28(OH)2] Br0.28·0.69 H2O was used as inorganic drug delivery system for anticancer therapy. Two different molecules were selected for being separately intercalated into ZnAl-HTlc: the anticancer drug norcantharidin, known for inducing cell cycle arrest at G2/M phase, and the tetra-sulfonated aluminum phthalocyanine, a


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photosensitizer used in anticancer photodynamic therapy (PDT). The obtained hybrid ZnAl-HTlc, were characterized in terms of X-ray powder diffraction pattern, thermogravimetric analysis, SEM microscopy, drug release profile, in vitro cytotoxicity, and ability to produce ROS and 1O2 upon light irradiation. Our results clearly indicate that the two selected compounds are efficiently intercalated within HTlc layers. Moreover, in vitro preliminary studies on a panel of cancer cells lines account for a greater cytotoxicity of the two drugs once loaded on HTlc either when administrated singularly or in combination. In addition, the analysis of the synergistic effect of the two formulations was evaluated by determining their combination index, which showed a greater cytotoxicity when using as a 1:2 ratio of AlPcS4@HTlc and NCTD@HTlc, respectively.


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INTRODUCTION

Combination chemotherapy regimens are often used in cancer therapy settings with the aim of attaining therapeutic synergism while overcoming resistance, possibly induced by the single-drug treatment.1 Neoplasms are very complex pathologies where the tumor mass grows uncontrollably involving genetic alterations and cellular abnormalities. Some problems associated with single-chemotherapy drug include the reduced accessibility of the drug to cancer tissues, which therefore requires higher doses that in turn lead to intolerable side toxicity and non-specific targeting. Furthermore, one of the main reason of chemotherapy failure is due to the ability of cancer cells to become resistant to numerous antitumor drugs by triggering escaping mechanisms able to inactivate the therapeutic effect.2,3 For this reason, in more aggressive cases, the clinical practice involves the use of combined therapies,4,5 as the simultaneous administration of different drugs able to tackle the tumor in a synergistic manner, increasing in some cases the expectation of recovery/survival.6–8 While successful at some extent and for specific type of cancers,9 combination therapy


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regimens are often selected on empirical basis and their efficacy is often hampered by the uncontrollable and different pharmacokinetic of the selected drugs, which might not allow to reach the required concentration able to induce the synergistic effect.

Recently, the use of nanotechnology has been widely considered in oncology as a valuable approach to deliver single or multiple therapeutic molecules at the injured site.10–12 Nanocarriers for cancer therapy are designed for promoting the in vivo penetration of biological barriers, such as epithelium and cell membrane, delivering the drug at the target site; moreover, they enable to preserve drugs activity, to prevent their degradation, improving their solubility and reducing their toxicity and side-effects.13–15

Among materials for designing anticancer nanocarriers, hydrotalcites (HTlcs) are very promising compounds due to their ease of synthesis, biocompatibility, pH-dependent stability and low toxicity to cells.16,17 In comparison to other drug delivery systems, e.g. gold, iron oxide, silica, and carbon nanotubes, HTlc layers dissolve without accumulating when the pH value is less than 5.5, making HTlc superior nanocarriers for controlled drug release under different pH conditions.18 Meanwhile, HTlc can effectively


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enhance the cellular uptake of intercalated drugs thank to the positively charged layer, which results in an improved passive targeting ability.19

HTlcs are described by the general formula [MII1-xMIIIx(OH)2]x+[An-x/n]x- ∙ mH2O, where MII is a divalent cation (Mg, Zn, Ni, Co or Cu), MIII is a trivalent cation (Al, Cr, Fe or Ga), An- is an anion with charge n and m is the molar amount of co-intercalated water. The x values, that represent the charge density of the positive layer and the anion exchange capacity, generally range between 0.2-0.4.20 Different organic, inorganic and metalloorganic anions may be intercalated in the interlayer region of HTlcs through an anion exchange reaction allowing to obtain a large number of hybrid compounds of potential interest in different field of applications.21,22 The present work focuses on the use of ZnAl-HTlc with formula [Zn0.72Al0.28(OH)2]Br0.28·0.69H2O, as inorganic drug delivery system for combined chemo and photo cancer treatment. Indeed, in addition to chemotherapy, alternative techniques such as photodynamic therapy (PDT), are increasingly considered for cancer treatment.23 PDT is a slightly invasive treatment in which a photoactive molecule, e.g. photosensitizer (PS), upon light irradiation at a


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specific wavelength, promotes the formation of singlet oxygen (1O2) and other reactive oxygen species (ROS) able to kill cancer cells.24,25 Interestingly, an increasing number of studies report the combination of PDT and chemotherapy as a promising strategy for improving anticancer therapeutic efficiency with minimized side effects. As an instance, Khdair et al.26 demonstrated the antitumor efficacy of doxorubicin in combination with the PS methylene blue to verify the nanoparticle-mediated combination treatment; Zhang et al.27 tested a chemo-photodynamic combination treatment with doxorubicin and chlorin e6 in different breast cancer cells (MDA-MB-231 and MCF-7); in addition Wang et al.28 studied an effective nanoscale therapeutic agent for combined chemophotodynamic therapy using doxorubicin and acryloyl meso-tetra(p-hydrosyphenyl) porphyrin, obtaining a synergistic killing effect on cancer cells, suppressing tumor development.

Among the different PS, phthalocyanines are promising candidates thanks to their tunable photophysical properties and more efficient generation of 1O2. In this regards, Kuznetsova et al., tested different phthalocyanines demonstrating that the nature of the


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central metal ion influences their photophysical properties; indeed, in tetra-sulphonated aluminum phthalocyanine (AlPcS4), the presence of aluminum increased the 1O2 production.29 Furthermore, AlPcS4 is characterized by a great absorption intensity in the near IR region of the spectrum allowing a deeper light penetration into tissues and improving the overall treatment effectiveness.30,31 In addition, our group demonstrated in an in vivo model of prostate cancer that AlPcS4 is able to significantly reduce the tumor growth and to induce histological changes like a higher level of apoptosis and vascular damage.31 Recently, Mei et al. successfully intercalated a Zn-Pc into HTlcs, followed by loading doxorubicin on the surface, in order to combine PDT and chemotherapy.32 For all these reasons we selected AlPcS4 as a PS to intercalate in ZnAl-HTlc.

Cantharidin, a natural compound isolated from the dried body of the blister beetle, is a strong and selective protein phosphatase 2A inhibitor, resulting in G2/M cell cycle arrest and apoptosis. As its clinical use is limited due to its gastrointestinal and genitourinary toxicity,33 norcantharidin (NCTD), a synthetic demethylated analogue, has emerged as a valuable alternative.34 As respect to cantharidin, NCTD maintains anticancer activity,


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while displaying reduced systemic and nephro-toxicity.35 NCTD has been widely used in China for the clinical treatment of several tumors, such as hepatocellular, breast, colon cancer and leukaemia.36–38 In particular, NCTD exerts its anticancer activity through different mechanisms, such as DNA synthesis arrest, caspases activation with consequent induction of cells apoptosis, mutation of proteins expression in cell cycle, inhibition of cell tumor proliferation and metastasis and activation of MAPK and protein kinase C.39,40 However, due to its poor solubility, alternative formulations of NCTD have been recently developed.41–44

In this work, we propose the preparation of hybrid ZnAl-HTlc compounds singularly intercalated with AlPcS4 (AlPcS4@HTlc) and NCTD (NCTD@HTlc) for the combined in

vitro treatment of a panel of cancer cells lines, with the aim of evaluating their possible synergic/additive effect on cancer cells killing.

EXPERIMENTAL SECTION

Materials


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ZnAl hydrotalcite (ZnAl-HTlc), with formula [Zn0.72Al0.28(OH)2]Br0.28 ∙ 0.69H2O, were obtained with the double microemulsion technique according to literature data.20

Intercalation of norcantharidin into HTlc (NCTD@HTlc)

The intercalation of NCTD into ZnAl-HTlc was performed by dispersing 150 mg of ZnAl-HTlc in 6.8 mL of a hydro-alcoholic (50% vol/vol) solution containing 57 mg of NCTD (0.34 mmol) and 0.7 mmol of NaOH. Br-/NCTD- molar ratio was 1:1. The reaction was performed under N2 atmosphere at 50 °C for 2 days. As reported for diclofenac intercalation,21 the obtained product was centrifuged with an Allegra 64R centrifuge (Beckman Coulter) for 5 minutes at 12000 rpm. The precipitate was washed 3 times with CO2-free water (2x30 mL) and dried at 60 °C overnight to give a white powder.

Intercalation of aluminum phtalocyanine into HTlc (AlPcS4@HTlc)

The intercalation of AlPcS4 into ZnAl-HTlc was performed by dispersing 150 mg of ZnAl-HTlc in 85 mL of an aqueous solution containing 76 mg of AlPcS4 (0.085 mmol) and 0.34 mmol of NaOH. Br-/AlPcS4- molar ratio was 4:1. The reaction was performed


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under N2 atmosphere at 50 °C for 2 days. As reported for diclofenac intercalation,21 the obtained product was centrifuged with an Allegra 64R centrifuge (Beckman Coulter) for 5 minutes at 12000 rpm. The precipitate was washed 3 times with CO2-free water (2x30 mL) and dried at 60 °C overnight to give a blue powder.

Characterization

Metal analyses were performed by Varian 700-ES series inductively Coupled PlasmaOptical Emission Spectrometers (ICP-OES).21 The ICP-OES measurements were performed in triplicate. The X-ray powder diffraction (XRPD) and thermogravimetric analyses (TGA) were performed by using previously reported instruments and conditions.20 TGA measurements were performed in triplicate. The morphology of the intercalated compounds was investigated with a Philips XL30 Scanning Electron Microscope (SEM), preparing the samples as previously reported.21 The diameters reported for NCTD@HTlc and AlPcS4@HTlc samples were the result of measurements on 30 nanoparticles. Stability studies in PBS containing 10% of FBS were performed by sedimentation test.17 In particular, AlPcS4@HTlc and NCTD@HTlc dispersions in water


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were sonicated for 10 minutes and then added to the stability medium achieving a final concentration of 1.25 mg/mL.

In vitro drug release study of NCTD from NCTD@HTlc

Release profiles of NCTD from HTlc were determined by through dialysis diffusion method, as previously described by Aluigi et al.45 A Specifically, a solution of the nanoparticles (1.25 mg/mL) dissolved in phosphate buffer (PBS), was inserted in a cellulose acetate membrane (cut off 12-14 kDa) and then immersed in 6 mL of PBS at pH 7.4 and 5.5, respectively, under moderate shaking at 37 °C. At different time points, every hour until seven for the first day and then every 24 h until seven days for a total of 168 h, the outer solution was taken (20 L) and fresh buffer (20 L) was added back. NCTD content released at different time points was analysed through LC-MS/MS technique using a Dyonex Ultimate 3000 HPLC coupled with a TS Quantum Access mass detector. Commercial LC-MS grade methanol and formic acid from Sigma-Aldrich were used without any further purification. A Millipore Milli-Q system was used to produce Ultrapure water (resistivity 18.2 MW/cm at 25 °C). In particular, LC was


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performed with a C-18 analytical column (Agilent SB-C18, 4.6 mm×150 mm, 5 m) by means of a linear gradient (1% formic acid water solution): (MeOH) from 50:50 to 20:80 at 0.5 mL/min flow rate (NCTD RT = 3.40 min). A negative ionization mode (ESI-) and a collision energy of 20 eV were set as mass spectrometry conditions and the transition of m/z from 185 (M-1) to 141 was selected for the quantification. The concentration was calculated by means of calibration curves. A stock solution of NCTD sodium salt was prepared to get calibration samples at 0.015, 0.5, 0.75 and 1.5 g/mL. A precision of RDS  15% was verified by three replicates. Regression equation of calibration curve was found linear in the range 0.015-1.5 g/mL. The response limit of detection was established as the first lowest point of the calibration curve, i.e. 0.015 g/mL (S/N  10). All experiments were performed in triplicate.

In vitro drug release study of AlPcS4 from AlPcS4@HTlc

Release profiles of AlPcS4 from HTlc were determined through dialysis diffusion method, as previously described by Aluigi et al.45 A Specifically, a solution of AlPcS4@HTlc (1.25 mg/mL) dispersed in PBS, was inserted in a cellulose acetate


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membrane (cut off 12-14 KDa), immersed in 6 mL of PBS and at pH 7.4 and pH 5.5, respectively, and moderate shacked at 37 °C. At different time points, every hour until seven for the first day and then every 24 h until seven days for a total of 168 h, the outer solution was taken (1 mL) and the fresh buffer was added back (1 mL). The AlPcS4 content at different time points was analyzed through an UV-Vis spectrophotometer Cary 100 (Agilent Technologies), considering the AlPcS4 absorption peak at 670 nm and comparing it with a previously acquired calibration curve. The PS release profiles were studied by curve-fitting analysis with different mathematical models, using Origin software.45 The experiment was performed in triplicate.

Singlet Oxygen detection

The generation of 1O2 was determined by using the chemical probe 9,10dimethylantracene (DMA). In particular, 100 L of AlPcS4@HTlc (1 mg/mL) and 600 L of DMA in dimethyl formamide (35 M) were inserted in a quartz cuvette and irradiated with a 300 W tungsten lamp at a distance of 40 cm for different irradiation times.


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Absorption spectra of the solution were recorded every minute for 10 minutes reading the peak decrease at 378 nm.

Reactive singlet oxygen species detection

ROS generation was determined by using the chemical probe 2,7dichlorodihydrofluorescein diacetate (H2DCFDA). In particular, H2DCFDA was dissolved in methanol obtaining a 1.1 mM solution. 2 mL of NaOH (0.01 M) were then added to 500 L of methanol solution and stirred for 30 min; afterwards 10 mL of PBS at 7.4 pH were added providing the ROS probe solution. Subsequently, 100 L of AlPcS4@HTlc in water were added to a cuvette containing 200 L of mQ-H2O, 500 L of PBS (pH 7.4) and 220 L of ROS probe as previously prepared. The solution was irradiated with a 300 W tungsten lamp at a distance of 40 cm for 5, 10, 15 minutes and the absorbance spectra were recorded at each time point with a UV-Vis spectrophotometer Cary 100 (Agilent Technologies), reading the peak increase at 500 nm.

In vitro cytotoxicity and cellular uptake studies


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HCT116 human colon carcinoma and HepG2 human hepatocytes cells were maintained in DMEM (Dulbecco Modified Eagle Medium); MCF7 and MDA-MB 231 human breast cancer cells were maintained in RPMI1640 (Roswell Park Memorial Institute 1640). The culture medium was supplemented with 10% fetal calf serum, 2 mM L-glutamine and 1% antibiotic mixture under standard culture conditions (95% O2 / 5% CO2 at 37 °C in a humidified atmosphere).46 Cell survival following exposure to all the compounds was evaluated using the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide]) assay, based on the reduction of MTT by living cells following exposure to all considered cytotoxic compounds.46,47 Briefly, cells were plated onto 96-well sterile plates and allowed to attach and grow for 48 h. NCTD and AlPcS4 were dissolved in DMSO and water respectively; AlPcS4@HTlc, NCTD@HTlc and nude HTlc nanoparticles were resuspended in water and mixed to obtain a uniform suspension. Complete medium was used to dilute all the solutions for cell treatment. Cells were treated with NCTD and AlPcS4, free or loaded onto HTlc, singularly and in a 1:2 combination ratio. The range of free drugs and drug@HTlc equivalent used was 0.001-250 M. After 24 h, the drug-containing medium was replaced by fresh PBS, and


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cells were irradiated under visible light for 2 h using a tungsten-halogen lamp (500 W; fluence of 158.4 J/cm2) as recently reported by Ballestri et al.48 At the end of irradiation, cells were incubated in the dark at 37 °C in drug-free medium and one day later MTT was added to each well (final concentration 0.4 mg/mL)46 and plates were incubated for 3 h at 37 °C. Cell viability was determined by measuring the absorbance using the Universal Microplate Reader (Bio-Teck-Instruments). The cytotoxic effect of the compounds, as free or loaded onto HTlc, was evaluated by calculating the drug concentration inhibiting cancer cell growth by 50 % (half maximal inhibitory concentration, IC50), based on non-linear regression analysis of dose-response data, performed using the Calcusyn software (Biosoft, Cambridge, UK).46 Results on cytotoxicity were analyzed for synergism by the median dose/combination index method developer by Chou and Talalay.49 According to this method, a combination index (C.I.) value 1.0 suggests an antagonistic interaction between the compounds. To assess AlPcS4 uptake, HCT116, HepG2, MCF7 and MDA-MB-231 cells were seeded onto six-well plates (3×105 cells/well) and exposed to 100 M AlPcS4 and AlPcS4@HTlc, alone or in


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combination with NCTD@HTlc (1:2) for 2 h. As recently reported,45 at the end of treatment, pictures of the cells were taken using an Olympus IX81 fluorescence microscope and cells were then rapidly washed with ice-cold PBS, detached with trypsin, re-suspended in ice-cold PBS and analyzed by flow cytometry, using a BectonDickinson FACS Calibur equipped with a 15 mW, 488 nm, air-cooled argon ion laser. The fluorescence emission was collected through a 575 nm band-pass filter in log mode and AlPcS4-fluorescence intensity was calculated from the flow cytometric profiles by the CellQuest Pro software (Becton Dickinson).45

RESULTS AND DISCUSSION

NCTD@HTlc and AlPcS4@HTlc synthesis and characterization

Nanostructured ZnAl-HTlc, with formula [Zn0.72Al0.28(OH)2]Br0.28 ∙ 0.69H2O were synthetized with the double microemulsion technique as previously reported.20 The occurrence of easily exchangeable bromide ions allows the functionalization of HTlcs with other guest species.21,50 To obtain the intercalation of NCTD and AlPcS4 into ZnAlHTlc, we prepared a hydro-alcoholic solution of the ring-opened dicarboxylic acid form


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of NCTD (0.05 M), obtained after treatment with NaOH, and an aqueous solution of AlPcS4 (0.001 M) (see experimental section), affording NCTD@HTlc and AlPcS4@HTlc, respectively (Figure 1A).

Figure 1. A) Schematic representation of AlPcS4@HTlc and NCTD@HTlc formation through anion exchange; B) XRD patterns of NCTD@HTlc (a) AlPcS4@HTlc (b) and pristine ZnAl-HTlc (c) nanoparticles; C) TGA curves of ZnAl-HTlc (yellow line), AlPcS4@HTlc (red line) and NCTD@HTlc (light blue line).


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In particular, to optimize the yield of NCTD intercalation, di-anionic NCTD concentration was varied between 0.005 M to 0.05 M (see Table S1 and Figure S1), indicating that 0.05 was the best molarity for the exchange reaction. Figure 1B shows the X-ray powder diffraction patterns of NCTD@HTlc, AlPcS4@HTlc and pristine ZnAlHTlc, showing that the interlayer distance increases from 8.06 Å for ZnAl-HTlc to 15.03 Å for NCTD@HTlc and to 23.1 Å for AlPcS4@HTlc, indicating the successful intercalation in the inorganic matrix.

The chemical composition of NCTD@HTlc and AlPcS4@HTlc samples was determined by thermogravimetric (TG) analysis and inductively coupled plasma-optical emission spectrometry (ICP-OES). The TG curve of sample NCTD@HTlc (Figure 1C, light blue line) shows a weight loss of 21% of the initial sample mass in the temperature range 80-250 °C, due to the loss of water adsorbed on particles surface and cointercalated within layers, and to the water removal upon dihydroxylation of the brucitic layers. In the same temperature range, for the AlPcS4@HTlc sample (Figure 1C, red line) also the decomposition of the carbonate ion occurs (weight loss of 18.5%). For


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both samples, in the temperature range between 250 and 600 °C, the weight loss is related to the removal of intercalated anions, that is of 17.4% for NCTD@HTlc (250450°C) and of 21.1% for NCTD@HTlc (250-600 °C). Considering the Zn/Al molar ratio and that ZnO and Al2O3 are formed at 800 °C, it was possible to determine the following composition for compounds intercalation: [Zn(0.670 ± 0.007)Al(0.330 ± 0.003)(OH)2](NCTD)(0.165 ± 0.002)·0.630

± 0.005 H2O (NCTD content of about 24% w/w) and [Zn(0.700 ± 0.007)Al(0.300 ±

0.003)(OH)2](AlPcS4)(0.0370 ± 0.0004)(CO3)(0.0760 ± 0.0008)·0.770

± 0.006 H2O (AlPcS4 content of

about 24% w/w).

The morphology and size of nanosized hybrid HTlcs were investigated by SEM analyses. In both cases (Figure 2C-D and 2E-F), as previously reported,21 the intercalation process led to the formation of large aggregates (around 1 m) constituted by the stacking of small hexagonal platelets (green arrows) with a diameter of 183 ± 61 nm for NCTD@HTlc and 215 ± 50 nm forAlPcS4@HTlc (Figure 2), which recall those of the original product (Figure 2A-B).51


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Figure 2. SEM analysis of ZnAl-HTlc (A,B), NCTD@HTlc (C,D) and AlPcS4@HTlc (E,F).

Stability studies on NCTD@HTlc and AlPcS4@HTlc were performed under mimed physiological conditions, i.e. PBS containing 10% FBS, demonstrating that, as previously reported for pristine ZnAl-HTlc,17 nanoparticles are very stable over time (Figure S2). In vitro drug release studies NCTD release from NCTD@HTlc. In vitro release study of NCTD@HTlc was performed both in PBS at pH 7.4, roughly simulating physiological conditions, and at pH 5.5 in order to simulate the acidic lysosomal conditions founded in cancerous cells.


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Figure 3. A) Release study of NCTD@HTlc in PBS at pH 7.4 (blue line) and pH 5.5 (bordeaux line). B) Release study of AlPcS4@HTlc in PBS at pH 7.4 (green line) and 5.5 (orange line). Results are the mean of three independent experiments ± SD.

As shown in Figure 3A, NCTD is similarly released under both conditions. In particular, while at the end of the observation time, i.e. 7 days, the difference in NCTD release under different pH conditions is not as evident, in the range between 10 to 100 hours, the NCTD release is higher at pH=5.5 than at pH= 7.4 (~10%).

AlPcS4 release from AlPcS4@HTlc. In vitro release study of AlPcS4 from HTlc was performed both in PBS at pH 7.4 and at pH 5.5 as earlier reported for NCTD@HTlc sample. Figure 3B shows that AlPcS4 is slightly released at both considered pH; in


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particular, the amount of AlPcS4 released from particles was 10% after 24 h and about 20% after seven days.

The slower release observed for AlPcS4 as compared to NCTD could be due to the higher charge density of AlPcS4 and to the higher affinity towards HTlc layers of SO3species with respect to the COO- groups of NCTD. However, it is worth noticing that, conversely to NCTD, AlPcS4 does not need to be released from particles in order to exert its biological activity, e.g. 1O2 and ROS production, thus the little release observed does not limit its efficacy. Moreover, this behaviour could be beneficial in in vivo environment since, until the sensitizer is retained on the particles, it is protected from aggregation and photo-bleaching phenomena, which would reduce its photo-toxicity.

Mathematical modeling of the release profiles

The release mechanism of NCTD and AlPcS4 from the matrices was investigated through different empirical and semi-empirical mathematical models (Tables S2 and S3), indicating that the diffusion mechanism predominates on the matrix swelling (a detailed description is reported in the supporting information section).52-54


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In order to evaluate the Fickian contribution on the release mechanism, the parameters F related to the releases of the two drugs in different pH environments, were plotted against the release times. As shown in Figure 4, the F value for NCTD release is higher than 1 at all the considered times, confirming that only the Fickian diffusion occurs during the release process. For AlPcS4 (Box of Figure 4), the contribution of Fickian diffusion is higher at pH 5.5 than at pH 7.4.

Figure 4. Fraction of drug released through Fickian diffusion.

ROS and 1O2 generation


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ROS generation efficiency of AlPcS4@HTlc was evaluated through the 2,7dichlorofluorescein (DCF) absorption peak at 500 nm. In the presence of ROS, the nonfluorescent molecule H2DCFDA is first hydrolyzed to 2,7-dichlorodihydrofluorescein (H2DCF) and then oxidized to the highly fluorescent species DCF. The solution containing AlPcS4@HTlc and a certain amount of H2DCF (see experimental part) was irradiated with a 300 W tungsten lamp for 5 min (fluence = 9.76 J/cm2), 15 min (fluence = 19.52 J/cm2) and 30 min (fluence = 29.29 J/cm2). As expected, the increase of the absorption at 500 nm indicates that ROS formation is dependent on irradiation time (Figure 5A), suggesting that even when loaded onto HTlc, AlPcS4 is able to induce ROS formation, while negligible ROS production was detected with empty HTlc (blank and empty HTlc curves are reported in supporting information, Figures S3A-B). 1O2 generation efficiency was calculated through the decrease of the absorption peak at 378 nm of DMA that is converted to the non-fluorescent endoperoxide form in the presence of 1O2.55 The solution containing AlPcS4@HTlc and DMA was irradiated with a 300 W tungsten lamp at 40 cm every minute for 10 min. As shown in Figure 5B, DMA absorbance gradually decreases with increasing the irradiation time from 0.55 to 0.43.


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Figure 5. A) Absorption spectra of 2,7-dichlorofluorescein (DCF) after different irradiation times of dichlorodihydrofluorescein (H2DCF) in the presence of AlPcS4@HTlc; B) absorption spectra of 9,10-dymethilantracene (DMA) in the presence of AlPcS4@HTlc.

In vitro cytotoxicity and cellular uptake

The effect of NCTD@HTlc and AlPcS4@HTlc on cell survival was evaluated both as single-drug administration (NCTD@HTlc or AlPcS4@HTlc) and as combination (AlPcS4@HTlc/NCTD@HTlc, 1:2 ) on four cancer cell lines, e.g. human colon carcinoma (HCT116), human hepatocellular carcinoma (HepG2), human breast cancer (MCF7) and human breast adenocarcinoma (MDA-MB 231). Importantly, at all


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considered concentrations, nude HTlc demonstrated to be safe for cells both in the dark and under light irradiation (data not shown).

Figure 6 shows the dose-response curves obtained following 24 h exposure to the four formulations (NCTD, NCTD@HTlc, AlPcS4 and AlPcS4@HTlc) as single agents, followed by 2 h irradiation (tungsten-halogen lamp 500 W; fluence of 158.4 J/cm2) and 24 h incubation in drug-free medium. In all cell lines, NCTD@HTlc and AlPcS4@HTlc produced a higher antiproliferative effect as compared to free compounds; statistical significance was confirmed by the analysis of IC50 values, calculated from the doseresponse curves and reported in Table 1. The intrinsic cytotoxicity of AlPcS4 and AlPcS4@HTlc was evaluated by omitting the irradiation step from the protocol and in all cases was found to be negligible (data not shown).


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Figure 6. Dose-response curves obtained in HCT116, HepG2, MCF7 and MDA-MB 231 by the MTT assay following 24 h treatment with NCTD and AlPcS4 free or loaded onto HTlc, 2 h irradiation under visible light and 24 h incubation in drug-free medium (mean ± S.E. of 3/6 independent experiments).

Table 1. IC50 values (M) obtained from the dose-response curves (mean ± S.E. of 3/6 independent experiments; * p

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