Amphiphilic Cyclodextrin Nanoassemblies with PDT

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Interface-Rich Materials and Assemblies

Tailored-Bodipy/amphiphilic cyclodextrin nanoassemblies with PDT effectiveness Roberto Zagami, Giuseppe Sortino, Enrico Caruso, Miryam Malacarne, Stefano Banfi, Salvatore Patanè, Luigi Monsu Scolaro, and Antonino Mazzaglia Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01049 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Tailored-Bodipy/amphiphilic cyclodextrin nanoassemblies with PDT effectiveness R. Zagami,a† G. Sortino,a† E. Caruso,b* M. C. Malacarne,b S. Banfi,b S. Patanè,c L. Monsù Scolaroc and A. Mazzagliaa* a

CNR-ISMN c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali

dell’Università di Messina, Viale F. Stagno d’Alcontres 31, 98166, Messina, Italy. b

Dipartimento di Biotecnologie e Scienze della Vita (DBSV) Università dell’Insubria, Via J.H.

Dunant 3, 21100 Varese (VA), Italy. c

Dipartimento di Scienze matematiche e informatiche, scienze fisiche e scienze della terra.

Università di Messina, Viale F. Stagno d’Alcontres, 31, 98166 Messina d

Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali dell’Università di

Messina Viale F. Stagno d’Alcontres 31, 98166, Messina, Italy and CIRCMSB, Unity of Messina.

ACS Paragon Plus Environment

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ABSTRACT

Amphiphilic cyclodextrins (aCD) are an intriguing class of carrier systems which, recently, have been proposed to deliver porphyrinoids and anticancer drugs or combined dose of both for dual therapeutic applications. The design of nanoassemblies based on aCD and photosensitisers (PSs) aims to preserve the PDT efficacy of PS, reducing the tendency of PS to self-aggregate, without affecting the quantum yield of singlet oxygen (1O2) production, and, not less importantly, minimizing dark toxicity and reducing photosensitization effects. With this idea in mind, in this paper, we focus on nanoassemblies between a non-ionic aCD (SC6OH) and halo-alkyl tailored iodinated BODIPY dye, a class of molecules which recently have been successfully proposed as a stimulating alternative to porphyrinoids for their high photodynamic efficacy. Nanoassemblies of BODIPY/aCD (BL01I@SC6OH) were prepared in different aqueous media by evaporation of mixed organic film of aCD and BODIPY, hydration and sonication. The nanostructures were characterized measuring their hydrodynamic diameter and ξ-potential, also evaluating their timestability in biological relevant media. Taking advantage of emissive properties of the notiodinated BODIPY analogue (BL01), nanoassemblies based on aCD and BL01 were investigated as model system to get insight on entanglement of BODIPY in the amphiphile in aqueous dispersion, pointing out that BODIPY is well-entrapped in monomeric form (τ ≅ 6.5 ns) within the colloidal carriers. Also morphology and fluorescence emission properties were elucidated after casting the solution on glass. BL01@SC6OH is easily detectable in cytoplasm of HCT116 cell lines, evidencing the remarkable intracellular penetration of this nanoassembly similarly to free BODIPY. On the same cell lines, the photo-dynamically active assembly BL01I/aCD shows


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toxicity upon irradiation. Despite the fact that free BL01I is more PDT active than its assembly, aCD can modulate the cell uptake of BODIPY, pointing out the potential of this system for in vivo PDT application.

INTRODUCTION The engineering of novel nano-oncologicals entrapping phototherapeutic agents, either as single molecule or in combination with other drugs, is a challenge aimed to improve outcomes in cancer treatment.1-3 In the field of phototherapeutics delivery, effort was carried out in Photodynamic Therapy (PDT) of tumors with the goal of increasing selectivity for the drug biodistribution into the diseased tissue and reduce skin photosensitivity which is, currently, a key limitation.4 Particularly, PDT is a cancer treatment modality which combines the use of a photoactivable drug (or phototherapeutic), named photosensitizer (PS), with light to cause local damage of the target tissue.5 Visible light of the appropriate wavelength, i.e. corresponding to an absorption band of the PS, is used to stimulate the photosensitizer (PS) into an electronically excited state (1PS). This latter can convert to its triplet state (3PS) by intersystem crossing (ISC). One important prerequisite in PDT is the high quantum yield for the transition 1PS → 3PS, then the triplet state energy is transferred to the ground state molecular oxygen to produce an excited species known as singlet oxygen (1O2). Singlet oxygen is a highly cytotoxic oxidizing species when it is photo-generated into the tissue, causing selective cell death in the irradiated region.6 Furthermore, PDT can activate immunological responses in cancer treatment7 as well as the destruction of the vasculature adjacent to the treated sites.8 Most of the first approved PSs (i.e. Photofrin®, mTHPC: Foscan®, and new ones, such as verteporfin (Visudine®), (chlorophyll-a derivative

(Radachlorin®),

bacteriopheophorbide-a

derivative

(Tookad®)

and


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pyropheophorbide-a derivative (HPPH), belong to the cyclic tetrapyrrolic class of molecules exhibiting relatively high affinity for tumor tissues, low intrinsic toxicity and intense absorption properties in the therapeutic window of the visible electromagnetic spectrum between 620 and 750 nm (red region).9 An alternative class of PSs, represented by non-porphyrin compounds based on a difluoro-boradiazaindacenes or boron-dipyrromethenes (BODIPY) core, has emerged over the last decade. BODIPY derivatives have been recognised as useful fluorophores in different biomedical applications, such as diagnostic probes,10-11 and photoactivable photherapeutics.12 BODIPYs can be obtained with high yield in a straightforward synthesis, showing high versatility towards further chemical modifications,

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high fluorescence quantum

yield (Φfl) and resistance to photobleaching.14-15 These latter two features are, however, a doubleedged sword: in fact, while photodynamic activity may benefit from a long half-life of photoactivatable molecules, this might be associated with persistent skin photosensitization. Furthermore, although a high quantum yields for fluorescence is fundamental when BODIPYs are exploited as biological probes, this characteristic should be limited when BODIPYs are engineered for PDT and the production of cell toxic reactive oxygen species must be maximized. The introduction of halogens (i.e. Br or I) at the BODIPY 2,6-pyrrolic position produces the internal heavy atom effect, promoting spin-orbit coupling, and then enhancing the ISC efficiency from 1PS to 3PS that guarantees the singlet oxygen production.16-17 In the recent past, some of us synthesized a series of BODIPY derivatives with significant potential in PDT and antimicrobial PDT.18-20 Furthermore, the selectivity of BODIPY for tumor cells can be accomplished by different strategies such as the decoration with targeting units,21-22 including inhibitor for kinase enzymes over-expressed by cancer cells23 or portions stimulating Ca2+ release in cell membranes.24

Furthermore,

the

versatility

of

BODIPY

skeleton

towards

chemical


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modifications25 allows the covalent binding with, for example, efficient NIR absorbing groups to achieve low-power density PDT,26 NO photodonors27 or aza-groups28 for combined phototherapies, and fluorophores for dual PDT and imaging treatment.29 Furthermore, coupling covalently BODIPY with biocompatible polymers30-31 or nanomaterials,32 assembling BODIPY moieties with host molecules to form photo-activable vesicles33 or loading BODIPY molecules into nanoparticles without34-36 or modified with surface receptor targeting groups,34,

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are

intriguing approaches to pursue a spatial-temporal control release and action. In the recent past, we have extensively developed supramolecular assemblies based on amphiphilic cyclodextrin (aCD) as nanocarriers for chemo-38-39 and/or photo-therapeutics sustained delivery40-42 by focusing both on photo-dependent43-45 and photo-independent effects.46 Moreover, self– assembled photoactivable vesicles based on phthalocyanines or squarines and aCD were recently proposed.47-49 As far as the light wavelength usable for the PSs excitation is concerned, BODIPYs with NIR absorbing moieties are generally used as PDT agents with light within therapeutic window. However green-light absorbing BODIPYs are designed as valid choice in multimodal approach, taking advantage of the less invasiveness of this wavelength in normal tissues around the tumor area.50-51 Along this direction, in this paper we propose nanoconstructs based on high biodegradable cyclodextrin38, 42 entrapping two novel synthesized BODIPYs: BL01I as potential PDT agent or BL01 as drug model and fluorescent dye for bio-imaging studies (see Scheme 3). As novel proof of concept, these double-faceted BODIPYs bear photoactivable moiety and an alkyl chain featuring a bromine atom on the farthest carbon which, in principle, can be used to further graft targeting groups and in perspective to achieve selective recognition sites on nanoassembly surface in biological environment. The length of the chain was selected to confer


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geometric similarities to the thioalkyl groups of aCD and to address hydrophobic interactions with aCD. Furthermore, recent literature proposed molecules bearing bromoalkyl groups to trigger cell death and anticancer effect52-53 Here, in particular, we only focus on PDT effects of BODIPY nanoconstructs, after assessing viability of cancer cells treated with free BODIPY in the dark at the investigated concentration. The final goal was to get highly dispersible nanoparticles in aqueous solution with low dye leakage, with enhanced photo-stability and satisfying photodynamic activity. The entanglement of the tailored BODIPYS in aqueous media containing non-ionic aCD was investigated by complementary techniques such a UV-Vis, steady-state fluorescence emission, dynamic light scattering (DLS) and ξ-potential measurements and fluorescence microscopy. Taking advantage of its fluorescence, we used the not-iodinated BL01 to focus on structural behavior of this typology of PS in aCD nanoassemblies, investigating the BODIPY/aCD interaction by time-resolved fluorescence emission. Stability in biological relevant media, phototoxicity of nanoassemblies vs free BODIPY on HCT116 cells and cell-uptake of nanoassemblies based on more emissive BL01 were also investigated. EXPERIMENTAL SECTION Synthesis of BODIPY derivatives. Synthesis of parent compounds. 4-(8-bromooctaoxy)benzaldehyde (1): 976 mg of 4hydroxybenzaldehyde (8 mmol), 8.4 g of Na2CO3 (80 mmol) and 180 mg of Bu4N+Cl- were dissolved in 30 mL of acetone and the solution was stirred at r.t. for 20 minutes. After this time 4.4 mL of 1,8-dibromooctane (24 mmol) were added. The reaction progress was monitored by TLC analysis (SiO2, CH2Cl2) and at complete consumption of the 4-hydroxybenzaldehyde (4 days) 100 mL of deionized water were added. The product was recovered by extraction with


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CH2Cl2 (3 x 30 mL), then the organic phase was dried over Na2SO4, filtered and evaporated to dryness. The raw material was purified by column chromatography (SiO2, CH2Cl2) affording 1.98 g (6.33 mmol, yield: 79.1%) of desired aldehyde as a pale yellow oil. C15H21BrO2 MW = 323.23 g/mol. 1H NMR (CDCl3) δ 1.39 (m, 4H, 2×CH2), 1.42 (m, 4H, 2×CH2), 1.82-1.90 (m, 4H, 2xCH2), 3.43 (t, 2H, 2×CH2-Br), 4.06 (t, 2H, CH2-O), 7.01 (d, 2H), 7.85 (d, 2H), 9.90 (s, 1H, CHO). 2,6-diiodo-1,3,5,7-tetramethyl-8-(4-(8-bromooctaoxy)phenyl)-4,4’-difluoroboradiazaindacene (BL01I): 484.8 mg of 4-(8-bromooctaoxy)benzaldehyde (1.5 mmol) and 340 µL of 2,4dimethylpyrrole (2.53 mmol) were dissolved in dry CH2Cl2 (30 mL) under N2 atmosphere, ten drops of TFA were added and the solution was stirred at RT overnight or for the time necessary to obtain the complete consumption of the aldehyde as determined by TLC analysis. Thereafter the dipyrromethane to dipyrromethene oxidation was achieved, in the same flask, by the addition of 397 mg of DDQ (1.75 mmol) and stirring continued for 30 min. The last step of the BODIPY synthesis requires the addition of Et3N (3 mL) and BF3.OEt2 (3 mL) to the reaction mixture to get the desired boronated final compound after 12h under stirring. The organic layer was subsequently washed two times with water, one with HCl 1 M solution and another two times with water; the organic solution was then dried over Na2SO4, filtered and evaporated to dryness. The raw material was purified by column chromatography (SiO2, petroleum ether-CH2Cl2, 3:7) affording 365 mg (0.687 mmol, yield: 46%) of the desired compound (BL01) as orange needles, mp > 300 °C. C27H34BBrF2N2O MW = 531.28; UV/vis (DCM): 501 nm (ε = ≅ 28200 M-1 cm-1); 1

H NMR (CDCl3) δ 1.27 (s, 6H, 2×CH3), 1.87 (m, 4H, 2×CH2), 2.16 (m, 8H, 4xCH2), 2.56 (s,

6H, 2xCH3), 3.44 (t, 2H, CH2-Br), 4.02 (t, 2H, CH2-O), 5.99 (s, 2H), 7.02 (d, 2H), 7.15 (d, 2H). The corresponding 2,6-diiodo compound (BL01I) was synthesised treating with I2 (0.460 mmol)


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and HIO3 (0.460 mmol) an ethanol solution of 122 mg of BL01 (0.230 mmol). This mixture was stirred for 15–18 h at RT and then washed with water and extracted three times with CH2Cl2; the organic phase was dried over Na2SO4 and evaporated to dryness. The crude product was purified by column chromatography (SiO2, petroleum ether-CH2Cl2, 1:1) affording 39 mg (0.05 mmol; yield: 22%) of 14 as red needles, mp > 300 °C; EA: calcd for C27H32BBrF2I2N2O C, 41.41; H, 4.12; N, 3.58; found: C, 41.58; H, 4.15; N, 3.59; UV/vis (DCM): 534 nm (ε = ≅ 41500 M-1 cm-1); 1

H NMR (CDCl3) δ 1.28 (s, 6H, 2×CH3), 1.42-1.56 (m, 8H, 2×CH2), 1.84-1.92 (m, 4H, 4xCH2),

2.66 (s, 6H, 2xCH3), 3.45 (t, 2H, CH2-Br), 4.04 (t, 2H, CH2-O), 7.04 (d, 2H), 7.14 (d, 2H) ppm (see figure S1A);

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C NMR (CDCl3) δ 159.70, 155.23, 143.17, 141.98, 131.89, 129.18, 126.89,

121.07, 115.32, 115.20, 115.08, 68.09, 33.93, 32.77, 29.22, 28.68, 28.08, 25.97, 14.57, 12.13 (see figure S1B). MS (ESI): M+ found: 782.96 calcd for C27H32BBrF2I2N2O 783.08; HPLC: retention time = 18’45’’ (99%), capacity factor, k = 2.57. Preparation of Nanoassemblies based on BODIPY and aCD. Nanoassemblies of BL01@SC6OH ([BL01] = 18 µM) or BL01I@SC6OH ([BL01I] = 20 µM) were prepared at 1:10 and 1:2 molar ratio, respectively, according to a reported procedure.41 Briefly, an organic film was obtained by slow evaporation of a mixed solution of SC6OH ([M33EO]= 3296.4; MALDI is reported in Figure S2) and BODIPY at the above-indicated molar ratios in dichloromethane (DCM). This film was hydrated with ultrapure water or PBS (10 mM phosphate buffer containing NaCl (137 mM) and KCl (2.7 mM) at pH 7.4) or NaCl (0.9 wt %) and sonication in ultrasonic bath for about 20 minutes. The unloaded BODIPY was separated by nanossemblies following slight centrifugation (5000 rpm) of the dispersions. BODIPY loading and entrapment efficiency. BODIPY loading into the NPs (drug loading) and entrapment efficiency percentages (EE %) were evaluated dissolving a known amount of each


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type of freeze-dried NPs (~ 1 mg) in 2 mL of DCM under magnetic stirring and analyzed by UV/vis spectroscopy. The actual drug loading and EE % were calculated, respectively, using the equations here reported: Drug Loading (%) = (amount of BODIPY in NPs/weighted amount of NPs) ×100

(1)

EE (%) = (amount of BODIPY in NPs/amount of BODIPY initially added to formulation) ×100 (2) Characterization of the nanoassemblies. Size and ζ-potential measurements. The mean diameter, width of distribution (polydispersity index, PDI) of the empty SC6OH, BL01@SC6OH and BL01I@SC6OH nanoassemblies and ζpotential were measured through Photon Correlation Spectroscopy (PCS) by a Zetasizer Nano ZS (Malvern Instrument, Malvern, U.K.). equipped with a He−Ne laser at a power P = 4.0 mW and λ = 633 nm. The measurements were performed at a 173° angle with respect to the incident beam at 25 ± 1 °C for each dispersion of SC6OH and BODIPY/SC6OH nanoassemblies using ultrapure water or a physiological solution (NaCl 0.9 wt %) or PBS (10 mM, pH 7.4) as 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. For ζ-potential measurements, empty SC6OH, BL01@SC6OH and BL01I@SC6OH nanoassemblies were dispersed in ultrapure water. The results are reported as the mean of three separate measurements on three different batches ± the standard deviation (SD). UV/vis spectroscopy. UV/vis spectra were obtained on a Hewlett-Packard (Agilent) model 8453 diode array spectrophotometer using 1 cm path length quartz cells. Extinction coefficient of


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BODIPY (ƐBODIPY(DCM)), and BODIPY@SC6OH (ƐBODIPY@SC6OH(PBS)), were determined by Lambert and Beer law in the range 1−50 µM. Steady-state fluorescence spectroscopy. Steady-state fluorescence measurements were performed on a Jasco model FP-750 spectrofluorimeter by using a 1 cm path length quartz cell. Stability studies. Stability studies were carried out in the dark by dissolving BL01@SC6OH and BL01I@SC6OH, respectively, in ultrapure water, PBS (10 mM, pH=7.4) or NaCl (0.9 wt %). All the dispersions were stored at 25 °C and monitored by UV/Vis and PCS along two weeks. ζpotential was measured along two weeks on the dispersions prepared in ultrapure water, stored at 25°C. Fluorescence images and morphological properties. The sample was prepared drying overnight a single drop of BL01@SC6OH dispersion in ultrapure water (BL01] = 0.36 µM and [SC6OH] = 3.6 µM) at room temperature using a glass cover slip as substrate. The fluorescence image has been acquired in scanning transmission mode exciting the sample with a solid-state laser working at 473 nm and focusing the light with a 100X SLWD Mytutoyo objective, NA 0.85. The laser power was kept well below a few tenths of microwatts to avoid any sample damage.54 The fluorescence was collected by a lens installed below the sample and coupled to an optical fiber. A couple of RazorEdge® ultrasteep long-pass edge filter cut off the laser photons and leave the fluorescence to reach the input window of an Hamamatzu miniaturized phototube. A lock-in technique was employed to improve the signal to noise ratio. The morphological properties of the sample have been investigated with AFM measurements using an NT-MDT NTEGRA Spectra microscope equipped with a V-tip working in semicontact mode. Photodynamic activity and photostability studies.


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Comparative Singlet-Oxygen generation measurements. The amount of produced 1O2 was determined by a standard method based on the bleaching reaction of p-nitrosoN,N’dimethylaniline (RNO).55 BL01I (50 µM) was prepared from DMSO stock-solution and diluted in PBS (10 mM, pH=7.4). BL01I@SC6OH ([BL01I] = 50 µM) dispersions were prepared from water stock-solution and diluted in PBS (10 mM, pH=7.4). Imidazole (10 mM) and RNO (50 µM) were added to BL01I and BL01I@SC6OH in PBS, so that the optical density at the excitation wavelength did not exceed the value 0.1, thus avoiding shielding effects. In a typical experiment, 2 mL of reaction mixture were poured into a quartz cuvette with a path length d = 1 cm, and exposed to a green LED source, at room temperature and for 1 h (power density 3.036 × 10-3 W/cm2). The decrease of the RNO absorbance was registered at λ = 440 nm every 2 min. The values of relative 1O2 quantum yield for a studied system (Φ∆) can be directly obtained from the different slopes (π) of the rate of bleaching (∆OD440 nm vs time) by using Φ∆ (Rose Bengal, RB) = 0.75 in physiological solution as a secondary standard56 according to the equation: Φ∆ = Φ∆(RB) π / π(RB) Photostablity of the BODIPY and BODIPY/aCD nanoassemblies. PS dispersions were prepared by dissolving BL01 and BL01I in DMSO and diluting in PBS. PS/aCD dispersions were prepared by diluting water stock dispersions of nanoassemblies with PBS. Concentration of PS was fixed at 10 µM. These solutions were irradiated, for 2 h at room temperature, with the green LED (fluence 21.8 J/cm2); during this period 1 mL sample was collected every 30 min and analysed with the UV/vis spectrophotometer. The photo-degradation rate was determined from the plot of the decrease in the absorbance intensity vs time (see Figure S10). The curves were fitted with an exponential equation from which the half-degradation time has been calculated.


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Biological studies. General. The human colon adenocarcinoma cell line HCT116 was obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle medium (DMEM, Mascia-Brunelli, Milano, Italy) supplemented with 10% fetal bovine serum, 1% glutamine and 0.5% antibiotic mixture (penicillin, streptomycin and neomycin), in standard culture conditions at 37 °C in a humidified 5% CO2 atmosphere. For the BODIPY used as reference, the solutions were generally prepared by dilution with PBS of a stock BL01 or BL01I solution prepared in DMSO at 1 mM concentration. For BL01@SC6OH and BL01I@SC6OH the dispersions were prepared in ultrapure water and diluted in PBS. The dilution of the stock BODIPY organic solution was made in order to get a non toxic concentration of the organic solvent in the cell cultures, i.e. below 1%. Cellular uptake. 1.5 x 106 cells were seeded in Petri dish and allowed to grow for 48 h prior to treatment with BL01, BL01I, BL01@SC6OH and BL01I@SC6OH by fixing BL01 and BL01I at 5.0 µM. After 24 h, cells were harvested by trypsinization, counted by burker chamber, centrifugated and resuspended in 1 mL of NaOH 0.1 M solution containing SDS 1%. The absorbance was recorded and the resulting concentration was obtained with a calibration curve of the PS in a cell lysate SDS solution. IntraCellular compartmentalization. Highly fluorescent BL01 and BL01@SC6OH systems were used in the experiments of PS detection inside the cells. Thus, HCT116 cells were seeded onto coverslips (10000 cells), allowed to grow for 48 h and subsequently treated with BL01 and BL01@SC6OH ([BL01] = 3 µM). After 24 h, cell attached to coverslips were washed three time in PBS and fixed in 3% paraformaldehyde for at least 10 min and then washed in PBS three


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times. Coverslips were mounted on microscope slides. Images were acquired using a LEICA TCS SP8 X confocal laser scanning microscope. Cytotoxic studies. The antiproliferative effect of each compound was assessed using the MTT assay.57 Briefly, 1.5×105 cells/mL were seeded onto 96-well plates and allowed to grow for 48 h prior to the treatment, for 4 and 24 h, with 100 µL of BODIPY solutions at different concentrations: free BL01 and BL01@SC6OH were tested in the range from 10 µM to 0.25 µM, whereas free BL01I and BL01I@SC6OH from 6.4 µM to 0.01 µM for 4 h of incubation and 1.6 µM to 0.001 µM for 24 h of incubation. The free BL01 and BL01I concentrations used in the dispersions coincide with the amount of BODIPY loaded with nanoassamblies. After 24 h, the BODIPY-containing medium was replaced by fresh PBS, and cells were irradiated under visible light for 2 h using a green LED (power density 3.036 x 10-3 W/cm2, fluence 21.8 J/cm2 from 490 to 560 nm). At the end of irradiation, cells were incubated in the dark for 24 h at 37 °C in drug-free medium then MTT was added to each well (final concentration 0.4 mg/mL) and left reacting for 3 h at 37 °C. The formazan crystals, formed through MTT metabolism by viable cells, were dissolved in DMSO and the optical densities were measured at 570 nm using a Universal Microplate Reader EL800 (Bio-Tek Instruments). Possible intrinsic cytotoxic effects (which were not photoinduced cell death) of the BODIPYs were assessed on control cultures kept in the dark and treated, as described above, with concentrations of BODIPY tenfold higher than those used in the PDT experiments.

RESULTS AND DISCUSSION


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The desired aldehyde for the BODIPY synthesis was obtained by deprotonation of the 4hydroxybenzaldehyde in acetone using sodium carbonate (Na2CO3) in the presence of 10% of tetrabuthylammoniumchloride, as phase-transfer agent. After 20 minutes at RT a three-fold quantity of 1,8-dibromoctane with respect to the aldehyde was added and the reaction kept at RT for four days. The pure aromatic aldehyde [4-(8-bromoctaoxy)benzaldehyde, 1 was then isolated after chromatography (SiO2, CH2Cl2), in 79% yields (Scheme 1).

Scheme 1. Synthesis of 4-(8-bromoctaoxy)benzaldehyde 1

The BL01 derivative was synthesized via acid catalysed condensation of the aromatic aldehyde with 2,4-dimethylpyrrole in the presence of catalytic amount of trifluoroacetic acid (TFA), following the general methods described by Akkaya and Liu.58-59 The disappearance of the aldehyde spot in the TLC analysis, fulfilled for the reaction control, indicates the formation of dipyrrolylmethane that was subsequently dehydrogenated to dipyrrolylmethene with 2,3dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) added in the same flask. At the end, the mixture was treated with BF3.Et2O and Et3N thus achieving the complete synthesis of the desired BODIPY. The compound was isolated as pure solid after a single chromatographic purification step. The 2,6-positions of BL01 were modified with the insertion of iodine atoms (Scheme 2) to promote the passage of the PS from the singlet to the triplet excited state following irradiation, thus increasing the singlet oxygen production rate. Regarding the iodination process, we found that the conditions reported in the literature (I2, HIO3, 60 °C, 20 min)60 cause a partial degradation of the dipyrrolylmethene skeleton, leading to extremely low yields in the desired


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iodinated boron-compounds. The successful recovery of the iodinated derivatives was obtained using a lower temperature (20–25 °C) and increasing the reaction time to 15–18 h. In the 1H NMR spectrum (see Figure S1A) it is possible to observe the para substituted system in the aromatic ring (dd at δ 7.04 and 7.14 ppm) and, because of the iodination, the disappearance of the singlet at δ 5.99 ppm, specific in the BL01 spectrum. 13C-NMR confirms the peculiar carbon resonances of BL01I (Figure S1B).

Scheme 2 Synthetic pathway to BL01 and BL01I.

Nanoassemblies based

on BL01 and

BL01I loaded SC6OH (BL01@SC6OH and

BL01I@SC6OH) were prepared by hydration of an organic film made of a mixture of aCD and BODIPY (BL01 or BL01I), followed by sonication, as previously reported.41 Mild centrifugation was necessary to purify nanoassemblies aqueous dispersions from unloaded and not-water soluble BODIPY. Scheme 3 sketches the formation of the nanoassemblies in aqueous dispersion from the above mentioned precursors.


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Scheme 3 Sketched view and molecular formulas of SC6OH, BODIPYs (BL01I or BL01) and their BODIPY@SC6OH nanoassembly.

Table 1 outlines the properties of both the BODIPY/aCD nanoassemblies, Values for unloaded aCD are reported in Table S1 for comparison.

Table 1. Overall properties of BL01@SC6OH and BL01I@SC6OH nanoassemblies (see Experimental section): Mean Hydrodynamic Diameter (DH) and Polydispersity Index (PDI) in ultrapure water, PBS (10 mM, pH 7.4) and in aqueous solution of NaCl (0.9 wt %), respectively (size distributions are reported in Figure S3), and ζ−Potential in ultrapure water (ζ). SD was calculated on three different batches. System

Dispersion

DH (nm ± SD)(%)a

PDI

ζ (mV ± SD)

medium

BL01@SC6OH

H20

PBS pH 7.4

Theoretical drug

245 + 45a (69)a

≤ 0.36

7 + 1a,b (31)a

≤ 0.28

203+ 41a (70)a

≤ 0.40

-

≤ 0.25

-

a,b

a

8 +1 (30)

-33 + 5

c

Actual

d

EE (%)

drug

loading (%)

loading (%)

7.39

6.54 + 0.20

88.5 + 2.7


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NaCl (0.9% wt)

250 + 26a (86)a a,b

BL01I@SC6OH

a

≤ 0.21

7 + 0.7 (14)

≤ 0.21

H2O

255 + 25a,b (100)a,b

≤ 0.20

PBS pH 7.4

220 + 40a,b (100)a,b

≤ 0.33

NaCl (0.9% wt)

a,b

a,b

210 + 15 (100)

-24 + 4

10.50

4.68 + 0.40

44.6 + 3.8

≤ 0.14

a

Size with corresponding Intensity % distribuition.b Size with corresponding number % distribution: in the presence of two families of nanoassemblies, number % for smaller nanoassemblies is ≅ 99.9 %. cActual loading is expressed as the amount of PS (mg) encapsulated per 100 mg of nanoassembly. dRatio between actual and theoretical loading x 100.

By considering the intensity % distribution, SC6OH forms in ultrapure water two families of nanoassemblies with DH of about 120 and 8 nm. These values agrees with our previous structural evidences.61 It is noteworthy that SC6OH utilized in this study has a higher average number of ethylene oxide chains (nEO ≅ 33 corresponding to the most probable peak, Figure S2), deriving from a more extensive anionic oligomerization of ethylene oxide, with respect to the non-ionic aCD carrier utilized in other reports;46 thus, in this case, we detect the presence of smaller selfaggregates with lower ζ and higher colloidal stability. DH increased remarkably in BODIPYloaded nanoassemblies in ultrapure water, albeit BL01@SC6OH showed a surface charge (ζ ∼ 35 mV) similar to the unloaded aCD. On the other hand, BL01I@SC6OH nanoassemblies exhibits a less negative ζ value (≅ -25 mV). The higher value of ζ for not-iodinated nanoassemblies vs iodinated suggested an increased tendency for BL01I to aggregate. Interestingly, all the BODIPY-loaded nanoassemblies in PBS at pH= 7.4 are in average smaller than unloaded cyclodextrin nanoparticles in the same medium. In the case of BL01I@SC6OH, a slight precipitation, which was not visually distinguishable, could take place, thus only the smaller nanoassemblies are stable in PBS. However, in all the cases, if we normalize for the number of nanoassemblies ( number % of distribution), the number of smaller nanoassemblies


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with corresponding negative ζ ( see Table 1) is predominant (≅ 99.9%), pointing out to the high stability of these small aggregates. Moreover, the reduced entrapment efficiency of BL01I in SC6OH, with respect to the not- iodinated analogue (EE% is 88.5 for BL01 in BL01@SC6OH vs 44.6 for BL01I in BL01I@SC6OH) could be correlated not only with the lower amount of aCD used in the preparation but also with the higher lipophilicity of the 2,6-iodinated BODIPYs with respect to those featuring only hydrogens. Indeed, by changing the amount of carrier (up to 1:10 BL01I/aCD molar ratio), aggregation phenomena were evident, thus, in order to minimize these latters, we opted to prepare BL01I@SC6OH nanoassemblies by using a lower carrier amount (at 1:2 molar ratio). Interaction studies by UV-Vis and Steady–stationary and Time-resolved fluorescence emission. UV/Vis and fluorescence emission were carried out to get insight on the interaction of the BODIPY in aCD carrier. The UV/Vis absorption spectrum of BL01 in DCM shows a band centered at 501 nm. BL01@SC6OH nanoassemblies dispersed in PBS show a UV/vis spectrum with a band centered at 503 nm, thus pointing out the complexation of BL01 with aCD (Figure 1A). Interestingly, only a very slight hypochromicity (of ≅ 2 %) in BL01@SC6OH with respect to BL01 was detected (ƐBL01@SC6OH(PBS) ≅ 27690 M-1 cm-1 vs ƐBL01(DCM) ≅ 28200 M-1 cm-1, see Figure S4), by confirming the excellent dispersibility of this BODIPY-loaded cyclodextrin in PBS. Steady-stationary fluorescence emission spectrum of BL01 in DCM (Figure 1B, trace a) exhibits a band with a maximum at 513 nm. After assembly with SC6OH in PBS, BL01 shows a band centered at 516 nm. This band has higher emission intensity and is broader vs that one of BL01 in DCM. These results are both presumably due to the effect of the colloidal aqueous environment (solvent effect) and to the interaction with amphiphilic molecules, confirming the


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suitability of aCD to entrap elevate cargo of BODIPY, even slightly improving the emissive properties.

Figure 1. UV/vis (A) (d = 1 cm), and fluorescence emission spectra (B) (λexc=466 nm) of BL01 in DCM (trace a) and BL01@SC6OH nanoassembly in PBS (10 mM, pH= 7.4) (trace b) at RT. In all the dispersions BL01 concentration was 16 µM.

Fluorescence emission properties of BL01 and BL01@SC6OH are summarized in the Table 2.

Table 2. Fluorescence emission properties of BL01 and BL01@SC6OH nanoassemblies.

a

System

Фfluo

rd

τ1 (ns)e

τ2 (ns)e

τ1A1 (%)e

τ2A2 (%)e

τR (ns)

BL01a

0.52c

≅0

1.8

4

4

96

0.6 ±0.2

BL01@SC6OHb

0.59c

≅0.15

2.8

6.5

4.5

95.5

0.5±0.2f; ≥ 20g

in DCM; bin PBS (10 mM, pH=7.4, r.t.); cvalues were determined as reported in experimental conditions

(see SI, eq. 2) and for comparison values see Ref 63; dstatic anisotropy (r) calculated at emission maximum (see SI, eq. 1) (λem=513 nm for BL01 and 516 nm for BL01@SC6OH); eFluorescence lifetimes were measured at λexc= 390 nm; Ai is the amplitude of the intensity decay; fτR1A1 is 4%. gτR2A2 is 96%.


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In agreement, the quantum yields of BL01 (DCM) is slightly lower than BL01-loaded SC6OH in PBS confirming especially the role of aCD to entrap and stabilize highly emissive BODIPY species (i.e. monomers, see infra in Fig 3). Furthermore, static anisotropy of BL01@SC6OH (ρ ≅ 0.15, see Table 2) is higher than in BL01 (ρ ∼ 0), in agreement with the entanglement of BODIPY in colloidal aCD. Moreover, the BL01@SC6OH nanoassembly was studied as a model because, actually, the loaded PS does not generate appreciable quantum yield of singlet oxygen (Ф∆(BL01@SC6OH) 20 ns), which was ascribed to the significant embedding of PS in the nanoassemblies.45

Figure 3. Fluorescence time decay (A) and time-resolved fluorescence anisotropy (B and C) (λexc = 390 nm) of BL01 in DCM (a) and BL01@SC6OH in PBS (10 mM, pH = 7.4) (b); [BL01] = 16 µM, RT.

When casted onto a glass surface and observed by optical microscopy, most of the particles exhibit fluorescence properties (Figure 4A). The morphology obtained through AFM (see Figure 4B) reveals that the surface is populated by a large number of almost spherical particles whose dimensions range from 300 nm to one micron, as indicated by the line profile reported in Figure 4C and acquired along the yellow vertical line depicted in Figure 4B.


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Figure 4. (A) Micro fluorescence of BL01@SC6OH casted on glass surface (see experimental methods). (B) Morphology of the squared area marked in figure A acquired by AFM technique in semicontact mode. Figure C is the line profile of one nanoparticle taken along the yellow vertical line marked in the bottom of the morphology.

All these results might agree with the model sketched in Scheme 3 in which plausibly BODIPY units are accommodated in the core of nanoassemblies probably by means of both selfinteractions among ω−bromohexyl chains. In our model, stacking between BODIPY moieties responsible for self-aggregation and reduction of photodynamic activity, should be strongly minimized. This idea is supported by the increase of the fluorescence lifetimes value (see Table 2) which are longer for BL01 loaded-aCD than in BL01 free in DCM. Furthermore, BODIPY chromophores seem to not interact with CD cavity as indicated by absence of induced circular dichroism in the region of BODIPY absorption (450-575 nm, data not shown). Stability studies. Stability studies of both BL01-and BL01I-loaded cyclodextrins were carried out in H2O, PBS and NaCl (0.9% wt), respectively, by monitoring the UV/Vis spectral changes and mean DH values along two weeks in the dark. Two weeks were selected as time window since within of this interval no incipient precipitation was observed for all the investigated system. The UV-spectrum of both freshly prepared dispersions of BL01@SC6OH and


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BL01I@SC6OH are shown in Figure 5. The peculiar band of BL01/aCD nanoassemblies was approximately unaltered by changing the nature of the dispersing medium (Figure 5A). On the other hand, BL01I@SC6OH spectrum shows that the band with maximum at 537 nm and a shoulder at ∼ 508 nm exhibits a reduced absorbance both in NaCl (0.9% wt) and in PBS dispersions ( Figure 5 B). This decrease was ascribed to the aggregation of nanoassemblies because of the increase of ionic strength.

Figure 5 UV/vis spectra of freshly prepared dispersions (RT) in different aqueous media (ultrapure water: solid line, NaCl (0.9 wt%): dotted line, PBS at pH = 7.4 dashed line) for BL01@SC6OH (A) and BL01I@SC6OH (B). In all the dispersions BL01 and BL01I concentration were 16 and 9 µM, respectively.

Figure 6 shows the changes of absorption spectra and mean DH vs time in ultrapure water for both BL01- and BL01I-loaded cyclodextrins. Within two weeks, the absorbance of the band at 503 nm slightly decrease (of about 14%) with respect to the freshly prepared dispersion (t=0) (Figure 6A). On the other hand, Figure 6B shows the decreasing of the absorption band at 536 nm of about 32% in two weeks. The average DH of BL01@SC6OH (Figure 6C) increase from ∼ 240 nm to ∼ 270 nm within 1 day, whereas it doubles after two weeks. Changes of ζ-potential (from ≅ -33 mV to ≅ -20 mV) witness the fair stability of these nanoassemblies within two weeks. Moreover BL01I@SC6OH forms aggregates of about 400 nm within 2 weeks, although


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only a slight change in the ζ-potential was observed (ranging between ∼ -25 and -20 mV). These results support that BL01@SC6OH and BL01I@SC6OH nanoassemblies are fairly stable in ultrapure water. The iodinated system shows a sharper decrease of the absorbance, probably ascribed to faster aggregation phenomena. However more stable and well-disperse aggregates are still present in water. Again, dispersions of both nanoassemblies in PBS and NaCl (0.9 wt %) were stored at 25°C. In these media, data (UV-Vis and DLS) pointed out that BL01@SC6OH and BL01I@SC6OH nanoassemblies are stable in 1-4 days range. (see Figure S8). Furthermore, by considering the gradual decrease of fluorescence emission vs time, our results suggest that the increase of size due to the aggregation of nanoassemblies is not causing any leakage of BODIPY from aCD, avoiding incipient precipitation of this PS in biologically relevant media.

Figure 6. UV spectra (A and B), mean DH (C and D, main populations only) and ξ-potential (E and F) vs time of BL01@SC6OH and BL01I@SC6OH (C and D) nanoassemblies in ultrapure water. In A and B spectra were acquired at t = 0, 1, 4, 7 and 14 days (follow the arrows). Nanoassemblies dispersions were


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stored at 25°C along the experimental time. In all the dispersions BL01 and BL01I concentration were 16 µM and 9 µM, respectively.

Comparative Singlet-Oxygen Generation Measurements and Photostability Studies. The production of 1O2 for each PS and PS/aCD nanoassemblies was indirectly determined measuring the disappearance of the absorption band (at 440 nm) of p-nitroso-N,N’-dimethylaniline (RNO). The experiments were carried out following a reported method, using PBS at pH=7.4 as solvent with a 50 µM initial concentration of RNO in the presence of imidazole (10 mM) and 50 µM of BODIPY.55 Irradiation was carried out with a green LED device. The choice of green light ensures a high level of electronic excitation of these PSs because of the good overlap between the λmax of BODIPYs absoption spectrum and the emission wavelength of the green LED, as well as with the absorbance profile of the standard dye. The relative rates of singlet oxygen (1O2) production were obtained comparing the kinetics of RNO degradation obtained with the BODIPY and with Rose Bengal as reference molecule (Figure S9). The results reported in Table 3 show that BL01 and BL01@SC6OH do not produce detectable amount of singlet oxygen, in agreement with findings established for the precursors and analogue derivatives of this BODIPY.19 On the other hand, both BL01I and BL01I@SC6OH are characterized by intense singlet oxygen production rates (ΦΔ 0.61 and 0.48, respectively), comparable with Rose Bengal (ΦΔ 0.75). In vivo PDT applications, the photostability of the photosensitizer plays a key role. Indeed, although a PS should be fairly stable to exert a good PDT action, an excessive stability could turn out to be highly phototoxic for a prolonged time after the treatment. Therefore, we have evaluated the photostability of the BODIPYs by irradiating a 10 µM solution in PBS for 120


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min, andrecording the absorbance at 30 min interval (Figure S10). The results show that the photostability of BL01 and BL01I is 44 and 49%, respectively, confirming the previously reported results.18 On the other hand the BODIPY-loaded aCD exhibits a higher photostability with respect to the same unloaded BODIPY (79% vs 44% and 65% vs 49% respectively for BL01 and BL01I). We are inclined to think that the shell of the nanostructure could exert a protective role on BODIPY photobleaching, catching the oxidizing species before they could reach the photosensitizer.

Table 3. Singlet oxygen generation quantum yield and photostability of the investigated systems.

a

BL01

BL01@SC6OH

BL01I

BL01I@SC6OH

∆1O2 a

-

-

0.61

0.48

Photostabilitya

44%

79%

49%

65%

green LED (power density 3.036 x 10-3 W/cm2, see Experimental section).

Photodynamic activity Assays. In order to evaluate the difference between the BODIPY vs BODIPY-loaded aCD, we have evaluated: i) the localization of the photosensitizers following cell uptake; ii) the intracellular compartmentalization of the PS-loaded aCD and PS free form; iii) the photodynamic activity on HCT116 cell line. To study the PSs localization, not-iodinated BODIPY, free or loaded in aCD, were used since these species are characterized by high quantum yield of fluorescence with respect to the iodinated compound. The cellular cultures were treated, over a period of 24 h of incubation, with 3 µM concentration of both PSs. An illustrative example of this analysis is shown in Figure 7. From this result, we can assess that no valuable differences could be found between BL01 and BL01@SC6OH intracellular compartmentalization of the PS in the cells and, as already reported for other PSs (porphyrins, chlorins, etc.),64 they are consistently localized in the cytosol district


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whereas none fluorescence could be observed in the nucleus. This result highlights the potential of BL01@SC6OH nanoassembly as diagnostic agent in bio-imaging investigations.

Figure 7. Confocal microscope images of HCT116 cells incubated with BL01 (panel A), and BL01@SC6OH nanoassemblies (panel B). [BL01] = 3 µM.

Cell uptake was assessed by considering the PS absorbance spectra of the cell lysate obtained after treatment of 1.5 x 106 HCT116 cell cultures with a 5.0 µM concentration of PS, both as nanoassemblies or in free form, for 4 or 24 h. As reported in Table 4, the free BODIPYs (BL01 and BL01I), after 4 h of incubation, show a twofold cell uptake compared to the BODIPYs nanoassemblies. Conversely, after 24 h of incubation, the difference between the free BODIPYs and the nano-assembled ones has decreased, thus indicating that BODIPY loaded in aCD needs a longer time to be uptaken in the cell than free BODIPY.


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Table 4: Cell uptake estimation of BODIPY upon HCT116 treatment with free BODIPY and BODIPY-loaded aCD Cell uptake estimation (nmol PS/106 cell)a

a

4 h of incubation

24 h of incubation

BL01

3.78

7.98

BL01@SC6OH

1.82

5.02

BL01I

3.61

7.23

BL01I@SC6OH

1.81

4.84

Values determined on HCT116 upon treatment, trypsinization/cell lysis and analyzed by UV/Vis: see

Experimental Section

The photodynamic activity obtained in HCT116 cells following exposure to the different PSs for 4 h and 24 h and irradiated with green LED device for 2 h are reported in Figure 8 as percentage of cell viability at different nM concentrations.

Figure 8. Photodynamic efficacy of free BL01, BL01I and BL01@SC6OH and BL01I@SC6OH nanoassemblies on HCT116 cell line, following 2 h irradiation with a Green LED device. A: activity of not-iodinated PSs after 24 h dark incubation; B: Activity of iodinated PSs after 4 h dark incubation; C: Activity of iodinated PSs after 24 h dark incubation.


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The intrinsic cytotoxicity of free PSs and the PS-loaded cyclodextrin nanoassemblies were assessed using concentrations 10-fold higher than those used for PDT experiments and omitting the irradiation step in the protocol. A negligible effect was observed in all cases (data not shown). At 24 h of incubation, BL01 is much less effective than the corresponding BL01I, in accordance with the previously reported results20 which assess how the presence of two iodine atoms on the BODIPY skeleton promotes the direct heavy atom effect, thus determining the increase of the singlet oxygen production and, consequently, a higher photodynamic effect. Our results evidence the effectiveness of SC6OH as PS carrier, making these highly lipophilic photosensitizers freely dispersible in water. In the absence of cyclodextrin, BODIPY solubilization in biological relevant medium can be achieved only in the presence of an organic vehicle such as DMSO or, alternatively, acetone. Unfortunately, these solvents exhibit some intrinsic toxicity and are not suitable in translational phase for approval in clinic. In this direction, it is important to evaluate the in vitro efficacy on cancer cell cultures of PS loaded in non-toxic carrier and perfectly dispersible in water. The comparison has been made between the free BODIPY, solubilized in a water/DMSO solution and the aqueous dispersion of the BODIPY@SC6OH nanoassemblies. From Figure 8C it is possible to observe how the BL01I@SC6OH shows a lower photodynamic efficacy than the BL01I both after 4 and 24 h of incubation. The highest difference is observed after 4 h of incubation and this result can be explained by considering the cell uptake data reported in Table 4 indicating how the BODIPY linked to the cyclodextrin requires a longer time compared to the free BODIPY to penetrate the cell. The higher in vitro photodynamic activity of BL01I compared to BL01@SC6OH can be explained by considering two experimental findings: the lower production of singlet oxygen


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(Table 3) and the lower cellular uptake (Table 4). In summary the data reported in this study seem to indicate a lower PDT efficacy of PS when embedded into the nanocarrier aCDs. However, the excellent solubilization effect with the formation of nanoparticles with suitable dimensions possibly exploiting the Enhanced Permeation Retention (EPR) effect in vivo should be considered. Indeed, along this direction, the treatment of solid tumors with drugs displays much more favourable conditions for preferential accumulation of nanoassemblies. With the prospect of a clinical application of these nanoparticles, we think that BL01I@SC6OH could be more advantageous as PDT agent that the free drug. Finally, the opportunity to introduce receptor targeting groups on BL01I@aCD, together with its bio-imaging properties, are intriguing to engineer novel multi-faceted nanoconstructs based on aCD and BODIPYs. CONCLUSIONS A new nanoassembly based on non ionic amphiphilic aCD and iodinated lipophilic BODIPY photosensitiser, characterized by the presence of a phenyl substituent in position 8, tailored with a ω-bromoalkyl chain, was successfully prepared in aqueous media. Highly dispersible nanoassembly entrapping an iodinated BODIPY (BL01I@SC6OH) was obtained. This system is fairly stable at room temperature in ultrapure water, PBS or physiological solution. An analogous nanosystem, based on not-iodinated BODIPY (BL01@SC6OH), was also prepared under the same conditions exploiting its fluorescence to investigate the aggregation behaviour of this family of PS into the nanoparticles. The presence of high percentage of monomers strongly embedded in the nanocarrier and the intracellular localization of our nanoassembly model, prompted the use of the iodinated BODIPY/aCD nanoassembly as PDT drugs. Indeed, the comparable values in the rate of singlet oxygen production of BL01I and BL01I@SC6OH and the higher photostability of BODIPY in the nanoassembly suggest the potential application of


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this nanosystem in photodynamic therapy. Possible drawbacks towards the use of the BODIPY/aCD nanoassembly come from the lower cyctotoxicity upon irradiation and lower cell uptake of this last as compared to free BL01I, probably due to the better cell permeability of free BODIPY. However, it is important to note that free lipophilic BODIPYs need the presence of organic solvents as vehicles to allow their solubility in aqueous environment and that the amount of these organic solvent (i.e. DMSO, acetone, ethanol) must be kept very low to avoid undesired side effects. Finally, and most importantly, it is conceivable that the in vivo application of this nanoassembly should be favoured by the well-known EPR effect, by which molecules of certain size preferentially accumulate in tumour than in normal tissues. ASSOCIATED CONTENTS Synthesis of BODIPY derivatives: General remarks; Preparation of Nanoassemblies based on BODIPY and aCD: General; Characterization of the nanoassembies: Steady-state fluorescence Fluorescence, Time resolved fluorescence; Photodynamic activity and photostability studies; 1H NMR and APT-13C NMR of BL01I ( Figure S1); MALDI-MS of SC6OH (Figure S2); Size distribution of SC6OH, BL01@SC6OH, and BL01I@SC6OH (Figure S3) and properties of SC6OH nanoassemblies (Table S1); Determination of extinction coefficients of BL01 and BL01@SC6OH (Figure S4); Determination of extinction coefficients of BL01I and BL01I@SC6OH (Figure S5); Fluorescence emission spectra of BL01, BL01@SC6OH, BL01I and BL01I@SC6OH (Figure S6); Stability studies by UV-Vis and DLS of BL01@SC6OH and BL01I@SC6OH in PBS (10 mM, pH= 7.4) (Figure S7); Stabilty studies by UV-Vis and DLS of BL01@SC6OH and BL01I@SC6OH in NaCl (0.9% w/t) (Figure S8); RNO degradation kinetics by singlet oxygen

of BL01I and BL01I@SC6OH (Figure S9); Photostability studies on PS

irradiated with the green LED for 120 min by UV-vis spectra (Figure S10).


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AUTHOR INFORMATION Corresponding Authors *Enrico Caruso, Department of Biotechnology and Life Sciences (DBSV). University of Insubria, Via J.H. Dunant 3, 21100 Varese (VA), Italy. Email: [email protected] *Antonino Mazzaglia, CNR-ISMN, Istituto per lo Studio dei Materiali Nanostrutturati, c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, V.le F. Stagno D’Alcontres 31 98166, Messina, Italy. Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. †These authors contributed equally. ACKNOWLEDGMENT The authors thank PON02_00665 (02_00355_2964193 HYPPOCRATES) and CNR (Project ISMN-CNR: Materials and Dispositives for Health and Life Quality) for financial support. REFERENCES 1. Kemp, J. A.; Shim, M. S.; Heo, C. Y.; Kwon, Y. J., “Combo” nanomedicine: Co-delivery of multi-modal therapeutics for efficient, targeted, and safe cancer therapy. Advanced Drug Delivery Reviews 2016, 98, 3-18. 2. Conte, C.; Ungaro, F.; Maglio, G.; Tirino, P.; Siracusano, G.; Sciortino, M. T.; Leone, N.; Palma, G.; Barbieri, A.; Arra, C.; Mazzaglia, A.; Quaglia, F., Biodegradable core-shell nanoassemblies for the delivery of docetaxel and Zn(II)-phthalocyanine inspired by combination therapy for cancer. J. Controlled Release 2013, 167 (1), 40-52. 3. Conte, C.; Ungaro, F.; Mazzaglia, A.; Quaglia, F., Photodynamic Therapy for Cancer: Principles, Clinical Applications, and Nanotechnological Approaches. In Nano-Oncologicals:


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SYNOPSIS


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Amphiphilic Cyclodextrin Nanoassemblies with PDT

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