Folate-Decorated Amphiphilic Cyclodextrins as Cell-Targeted

Article Cite This: Biomacromolecules 2019, 20, 2530−2544

pubs.acs.org/Biomac

Folate-Decorated Amphiphilic Cyclodextrins as Cell-Targeted Nanophototherapeutics Roberto Zagami,†,⊥ Valentina Rapozzi,‡,⊥ Anna Piperno,§,⊥ Angela Scala,§ Claudia Triolo,∥ Mariachiara Trapani,† Luigi E. Xodo,‡ Luigi Monsù Scolaro,§ and Antonino Mazzaglia*,†

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†

CNR-ISMN, Istituto per lo Studio dei Materiali Nanostrutturati c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali dell’ Università di Messina, Viale F. Stagno d’Alcontres 31, Messina 98166, Italy ‡ Dipartimento di Area Medica, Università di Udine, P.le Kolbe 4, Udine 33100, Italy § Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università di Messina, Viale F. Stagno d’Alcontres 31, Messina 98166, Italy ∥ Dipartimento di Scienze Matematiche e Informatiche, Scienze Fisiche e Scienze della Terra, Università di Messina, Viale F. Stagno d’Alcontres, 31, 98166 Messina, Italy S Supporting Information *

ABSTRACT: Nowadays, active targeting of nanotherapeutics is a challenging issue. Here, we propose a rational design of a ternary nanoassembly (SAP) composed of nonionic amphiphilic β-cyclodextrins (amphiphilic CD) incorporating pheophorbide (Pheo) as a phototherapeutic and an adamantanyl-folic acid conjugate (Ada-FA) to target tumor cells overexpressing α-folate receptor (FR-α(+)). Dynamic light scattering and ζ-potential pointed out the presence of nanoassemblies bearing a negative surface charge (ζ = −51 mV). Morphology of SAP was investigated by atomic force microscopy and microphotoluminescence, indicating the presence of highly emissive near-spherical assemblies of about 280 nm in size. Complementary spectroscopic techniques such as ROESY-NMR, UV/vis and steady-state fluorescence revealed that the folic acid protrudes out of amphiphilic CD rims, prone for recognition with FRα. Pheo was strongly loaded in the nanoassembly mostly in monomeric form, thus generating singlet oxygen (1O2) and consequentely showing phototherapeutic action. SAP remained stable until 2 weeks in aqueous solutions. Stability studies in biologically relevant media pointed out the ability of SAP to interact with serum proteins by means of the oligoethylenglycole fringe, without destabilization. Release experiments demonstrated the sustained release of Pheo from SAP in environments mimiking physiological conditions (∼20% within 1 week), plausibly suggesting low Pheo leaking and high integrity of the assembly within 24 h, time spent on average to reach the target sites. Cellular uptake of SAP was confirmed by confocal microscopy, pointing out that SAP was internalized into the tumoral cells expressing FR-α more efficiently than SP. SAP showed improved phototoxicity in human breast MCF-7 cancer cells FR-α(+) (IC50 = 270 nM) with respect to human prostate carcinoma PC3 cells (IC50 = 700 nM) that express a low level of that receptor (FR-α(−)). Finally, an improved phototoxicity in FR-α(+) MCF-7 cells (IC50 = 270 nM) was assessed after treatment with SAP vs SP (IC50 = 600 nM) which was designed without Ada-FA as a targeting unit.

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apeutics5 and diagnostics6 through endocytosis mediated by cell-cycle dependent expression of the α-folate receptor (FRα).7 Different typologies of folate-tailored nanoconstructs based on polymers,8 also possessing acid-labile9 or redox sensitive groups,10 on metal organic frameworks,11 nanoparticles combined with multiwalled carbon nanotubes,12 or with a polymeric core,13 nanocrystals,14 gold,15 and inorganic nanoentities16 have been recently developed. In the design of FR-α targeted nanoplatforms with longer circulation time in

INTRODUCTION

Nowadays, the design of novel multifaceted nano-oncologicals for active targeting of therapeutics and imaging agents is a serious challenge.1 Actually, the majority of FDA-approved nanomedicines rely on passive targeting via the enhanced permeability and retention (EPR) effect and only a few examples which employ active targeting approaches are in clinical phase for the treatment of solid tumors.2 Reproducible synthesis, proper chemical-physical characterization, and adequate knowledge of toxicity and events at the nanobiointerface in vitro and in vivo could yield faster the clinical translation of these nanodrugs.3,4 Folate coupling is one of the main investigated strategies to achieve tumor-targeted nanother© 2019 American Chemical Society

Received: February 28, 2019 Revised: June 1, 2019 Published: June 7, 2019 2530

DOI: 10.1021/acs.biomac.9b00306 Biomacromolecules 2019, 20, 2530−2544

Article

Biomacromolecules Scheme 1. Sketched View of SAP Formation from S, Ada-FA, and Pheo Components

theranostic hybrid functionalities50−54 or upconversion nanocomposites55,56 were proposed as promising candidates for increased PDT efficacy and/or optimized diagnostic properties. Pheophorbide-A (Pheo) is a chlorophyll catabolite with a relatively high 1O2 quantum yield and a strong PDT activity both in vitro or in vivo.57 Conjugation of Pheo or its derivatives to polymers,58 amphiphilic moieties,59,60 or peptides,61,62 or encapsulating this PS into nanocarries modified with receptor targeting groups (i.e., FR-α),63 improved the selectivity toward cancer cells.64 Along this direction and considering the great chance offered by CD nanoassemblies as drug delivery systems,28,65 in this paper we designed novel nanophototherapeutics composed of amphiphilic CD (nonionic amphiphilic βCD SC6OH (S)) decorated with FA. A noncovalent functionalization of the CD scaffold was carried out by proper fine-tuning at the nanoscopic level of the components assembly, thus controlling the loading and release properties of entrapped PS and its biological performance. Therefore, the targeted S@Ada-FA (SA) was obtained by supramolecular inclusion of the adamantyl group (Ada) conjugated with FA by an oligoethyleneglicole chain of length comparable to the external PEG fringe of amphiphilic CD. Pheo was noncovalently entrapped in the ternary (S@Ada-FA/Pheo (SAP, Scheme 1)) and in the binary (S/Pheo (SP)) nanoassembly, respectively. Rotating-frame Overhauser enhancement spectroscopy nuclear magnetic resonance (ROESY-NMR) experiments were carried out to elucidate the interaction of Ada with the CD cavity and the arrangement of FA. Electronic and emission properties, morphology, size, and surface charge of SAP and SP were investigated by UV/vis, dynamic light scattering (DLS), ζ-potential measurements, atomic force microscopy (AFM), and microphotoluminescence. Studies of release kinetics, drug and FA loading, stability in biologically relevant media, cell uptake and phototoxicity of SAP vs SP on cells overexpressing FR-α (breast cancer MCF-7, FR-α (+)) and cells down-expressing FR-α (prostate cancer PC3, FR-α (−)) were carried out to demonstrate the high selectivity of FA-decorated nanoconstructs toward FR-α (+)cells.

the body, a proper compromise between length and density of a polyethylene glycol (PEG) chain on the nanocarrier surface (i.e., short PEG 1−2 kDa) is highly desirable to minimize the absorption of medium protein components17 and/or the selfentangling of longer PEG chains (>2 kDa),18 which could compromise the exposition of folate to cell membrane receptors.19 Among various approaches, complexes based on cyclodextrins (CDs), macrocyclic oligosaccharides generally composed of six, seven, or eight D-glucopyranose units linked by α-(1,4) bonds (denoted α-CD, β-CD, and γ-CD, respectively) decorated with folic acid or folate (FA) are promising targeted drug delivery systems.20 FA-appended CDs have been obtained by supramolecular strategies21 or by covalently linking FA to the external rim of CD,22 leading to efficacious therapeutic constructs23 conjugating24 or entrapping anticancer drugs.19,25,26 FA-decorated nanoparticles based on polymers/CD27 or on amphiphilic CD were developed,28 the latter proposed for breast cancer treatment29 or therapeutic gene delivery30 or silencing on prostate cancer cells.31 Within our ongoing research on novel nano-oncologicals, our interest was focused on the design of aCD nanoassemblies for the delivery of anticancer agents32,33 and/or photosensitizers (PSs)34−36 for chemotherapy and photodynamic therapy (PDT) alone or following a dual drugs treatment.37 Modified CDs and nanoassemblies based on amphiphilic CDs were proposed as containers to bind PSs by host−guest interaction for applications in photodynamic therapy (PDT).38−40 PDT relies with combination of PS, light, and oxygen generating, among other, radical oxygen species (ROS) and singlet oxygen (1O2). Phototoxicity of PS can be reduced by its low solubility and tendency to self-aggregation which can decrease the 1O2 quantum yield. These drawbacks coupled with low selectivity for a pathological targeted area and prolonged systemic photosensitization, until now, have limited the applicability of PDT. Therefore, targeted PDT by entrapping or covalently linking PS in suitable nanocontainers, conjugated with tumortargeting ligands, could be a valid complementary approach to chemotherapy in the treatment of solid tumors.41 Multifaceted FA-decorated nano-photo-platforms based on polymers,42−44 proteins,45 liposomes,46−48 hydrogels49 without or with 2531


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Steady State and Time-Resolved Fluorescence Spectroscopy. Steady-state fluorescence measurements were performed on a Jasco model FP-750 spectrofluorimeter at r.t. ≅ 25 °C. Fluorescence emission spectra were not normalized by extinction at excitation wavelength. Time resolved fluorescence emission measurements were performed on a Jobin Yvon-Spex Fluoromax 4 spectrofluorimeter using time-correlated single-photon counting technique. A NanoLED (λ = 390 nm) was used as the excitation source. Microphotoluminescence and Morphology. AFM images of the sample surface were acquired with a NT-MDT NTEGRA Spectra microscope working in semicontact mode. Microphotoluminescence (micro PL) measurements were acquired in reflection mode at normal incidence. As the exciting source, we used a solid-state laser at λexc = 470 nm coupled to NT-MDT NTEGRA Spectra and focused onto the sample by a 100× objective (Mitutoyo, NA = 0.70). The same objective was used to collect the fluorescence signal that was analyzed by a monocromator (SOL Instruments) and detected by a cooled CCD Camera (Andor IDus).36 The sample was prepared drying overnight at r.t. a single drop of SAP dispersion in ultrapure water containing 0.045 μM entrapped Pheo using a glass coverslip as the substrate. SAP dispersion was obtained by dilution (∼1:150) of stock aqueous dispersion prepared at [S]:[Ada-FA]:[Pheo] 2.5:1:1 ([S] = 20 μM; [Pheo] = 8 μM; [Ada-FA] = 8 μM). Loading and Entrapment Efficiency of Pheo and Ada-FA within the Nanossemblies. Pheo actual loading inside nanoassemblies and entrapment efficiency percentage (EE %) were evaluated by dissolving a known amount of each type of freezedried nanassembly (∼1 mg) in 2 mL of DCM under magnetic stirring and analyzed by UV/vis spectrophotometry. A calibration curve for Pheo in DCM was performed in the concentration range 0.59−14.22 μg/mL. Ada-FA actual loading inside nanoassemblies and EE % were evaluated by dissolving a weighted amount of each type of freezedried nanoassembly (∼1 mg) in 2 mL of MeOH under magnetic stirring and analyzed by UV/vis spectrophotometry. A calibration curve for Ada-FA in MeOH was performed in the concentration range 1−50 μM. The drug loading and EE% were calculated, using the following equations, respectively:

EXPERIMENTAL SECTION

Materials. Heptakis(2-O-(oligoethylene glycol)-6-hexylthio)-βCD (SC6OH, S) with nEO = 3−4, corresponding to exact masses with 28−35 units of ethylene oxide (EO); [M33EO] = 3296.4) was synthesized and characterized according to the general procedures.36,66 Pheophorbide-A (Pheo, MW = 592.68) was purchased from Aurogene (Roma, Italy). Folic acid (FA), polyoxyethylene sorbitan monoleate (Tween 80), and all the reagents and solvents (analytical grade) were purchased from Sigma-Aldrich (Milano, Italy). All the dispersions used for nanoassemblies preparation and spectroscopic characterizations were prepared in ultrapure water (Galenica Senese, Siena, Italy) or in 10 mM phosphate buffer containing NaCl (137 mM) and KCl (2.7 mM) at pH 7.4 (PBS) at room temperature (r.t ≅ 25 °C). Interaction Studies by NMR. NMR spectra were recorded on a Varian 500 MHz spectrometer at r.t. ≅ 25 °C. The chemical shifts are expressed in ppm downfield from tetramethylsilane (TMS). The signals assignment of Ada-FA was determined by heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), and correlation spectroscopy (COSY) 2D NMR experiments. Samples were prepared as follows: Ada-FA (5 mg) was dissolved in DMSO-d6 (1 mL) under sonication; the S/Ada-FA complex for 1H NMR was prepared in D2O at 1.8/1 molar ratio ([S] = 2 mM, [Ada-FA] = 1.1 mM); the S/Ada-FA complex for ROESY experiments was prepared in D2O at 1/0.9 molar ratio ([S] = 2 mM, [Ada-FA] = 1.8 mM). Nanoassemblies Preparation. SAP and the analogues without Ada-FA (SP) or without Pheo (SA) were prepared at [S]:[Ada-FA]: [Pheo] 2.5:1:1 (SAP) and 2.5:1 (SP and SA) molar ratios, respectively ([S] = 200 μM; [Pheo] = 80 μM; [Ada-FA] = 80 μM) according to our previously reported procedure.36,67 Samples for spectroscopic measurement (DLS and ζ-potential, UV/vis, and fluorescence) were prepared by using components at molar concentrations 10 times lower. Briefly, S (1.3 mg) was dissolved in dichloromethane (DCM) and the solution was evaporated overnight to form a thin film. This solution was hydrated with ultrapure water or PBS at room temperature and sonicated in ultrasonic bath for about 20 min. SP was prepared by the hydration of a thin organic film of Pheo (0.1 mg) with the previous water or PBS dispersion of S at 50 °C followed by 20 min of sonication in an ultrasonic bath. For the preparation of SAP, an organic film of S@Ada-FA (SA) was obtained by evaporation of a mixed organic solution of S (1.3 mg) in DCM and Ada-FA (0.12 mg) in MeOH. This mixture was hydrated with water or PBS at room temperature and later sonicated for 20 min. Finally, a thin organic film of Pheo (0.1 mg) was hydrated with the previous water or PBS dispersion of SA at 50 °C followed by 20 min of sonication in an ultrasonic bath. The unloaded Ada-FA (in SA and SAP dispersions) or Pheo (in SP and SAP) was separated by slight centrifugation (6000 rpm) for 20 min. The supernatants were recovered and freeze-dried to get the solid cottony nanoparticulate. Size and ζ-Potential of NPs. The mean diameter and width of distribution (polydispersity index, PDI) of the empty S, and SA, SP and SAP were measured by photon correlation spectroscopy (PCS) by a Zetasizer Nano ZS (Malvern Instrument, Malvern, U.K.) utilizing a noninvasive 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. The deconvolution of the measured correlation curve to an intensity size distribution was achieved by using a non-negative least-squares algorithm. The ζ-potential values were determined using a Zetasizer Nano ZS Malvern Instrument equipped with a He−Ne laser at a power P = 4.0 mW and λ = 633 nm. UV/vis Spectroscopy. UV/vis spectra were obtained on a Agilent model 8453 diode array spectrophotometer using 0.1−1 cm path length quartz cells at r.t. ≅ 25 °C. A UV/vis neutral filter was located on the observation path made on the diode array setup in order to avoid the formation of hydrochloric acid produced at the interface between the light-exposed silica wall and the bulk solvent when dichloromethane was used as the solvent.68

Drug actual loading (%) = (amount of Pheo or Ada‐FA in nanoassembly /weighted amount of nanoassembly) × 100

(5)

EE (%) = (amount of Pheo or Ada‐FA in the nanoassembly /amount of Pheo or Ada‐FA initially added to formulation) × 100

(6)

Stability Studies. Stability studies were carried out by UV/vis and DLS vs time. SAP was incubated up to 2 weeks at 25 °C in different biological media: (i) ultrapure water, (ii) 0.9 wt % NaCl aqueous solution, (iii) PBS at pH 7.4, (iv) PBS with 2 wt % of human serum albumin (HSA), and (v) PBS with 2 wt % of fetal bovine serum (FBS). ζ-Potential was measured vs time on SP and SAP dispersed in ultrapure water. Each dispersion was kept in a cuvette, thermostated at 25 °C, and analyzed at r.t. in triplicate. The results are reported as the mean of three separate measurements on three different batches ± the standard deviation (SD). Comparative Singlet-Oxygen Generation Measurements and Photostability Studies. The amount of 1O2 produced was determined by a standard method based on the bleaching reaction of p-nitroso-N,N′-dimethylaniline (RNO).69,70 The tested sensitizers (SP and SAP, [Pheo] = 70 μM) were dissolved in PBS (10 mM, pH 7.4) with imidazole (10 mM) and RNO (50 μM), so that the optical density at the excitation wavelength did not exceed the value 0.1, to avoid shielding effects. In a typical experiment, 2 mL of reaction mixture was poured into a quartz cuvette with a path length d = 0.3 cm, and exposed to a red-LED homemade apparatus (λ = 660 nm, power density ∼ 2.6 mW/cm2) for different times (from 0 to 90 min). ) can be The values of 1O2 quantum yield for a studied system (φsystem Δ 2532


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Biomacromolecules Scheme 2. Synthetic Strategy for the Preparation of Ada-FA

directly obtained from the different slopes (πsystem) of the rate of = 0.59 in ethanol71 as a bleaching (ΔA440 nm vs time) by using φPheo Δ secondary standard according to the following equation:

composition in order to evaluate the influence of FA and serum proteins contained in the medium. Confocal Microscopy Studies. MCF-7 and PC3 cells were seeded on 19 mm coverslips in a 6-well plate at a density of 3 × 105 and 4 × 105 cells, respectively. The cells were treated with Pheo, SAP, and SP ([Pheo] = 2 μM) and incubated in the dark for 3 h in a medium without serum. After this time the glasses were prepared. The cells were washed twice with PBS and then fixed with 3% paraformaldehyde (PFA) in PBS for 20 min. After washing with 0.1 M glycine, containing 0.02% sodium azide in PBS to remove PFA, and Triton X-100 (0.1% in PBS), the glasses were treated with Hoechst in order to stain the nuclei of the cells. The analyses of the glasses was performed using a Leica TCS SP8 (Leica Microsystems, Heidelberg, Germany) confocal system equipped with a 405 nm diode laser and a tunable super continuum white light laser (λexc = 570 nm, 580 nm ≤ λem ≤ 778 nm). The uptake of the nanoassemblies was measured monitoring the red fluorescence between 580 and 778 nm emitted by Pheo upon excitation at 570 nm. Photodynamic Treatment in Vitro. MCF-7 and PC3 cells were seeded at a density of 7 × 103 cells into a 96-well plate. The day after, they were incubated in the dark for 3 h with different nanoassembly concentrations and then irradiated with a red LED apparatus (A.P.E. Research, Basovizza TS, λ = 660 nm). This instrument is based on light-emitted dose (LED) technology for evaluating the effects of photosensitizers. An optical 24 LED array was projected and developed to illuminate cells in standard 24-well or 96-well cell culture plates, under rigorous intensity control: irradiance intensity was adjustable (ranging from 4.5 to 140 mW/cm2) and the energy densities (fluencies) were regulated by the time period of LED irradiation. In our experiments of phototoxicity a power density of 2.6 mW/cm2 for 6 min (fluence of 0.9 J/cm2) was used. 24 h after light irradiation a cell proliferation assay was determined by the resazurin test following the manufacturer’s instructions (Sigma-Aldrich, Milan, Italy). The values were obtained by a plate-spectrofluorimeter (EnSpire 2300 Multilabel reader PerkinElmer, Finland). Annexin V-Propidium Iodide Assay. Apoptosis was assessed by annexin V, a protein that binds to phosphatidylserine residues, which are exposed on the cell surface of apoptotic cells. MCF-7 and PC3 cells were seeded in a 12-well plate at density of 1.5 × 105 and 1.8 × 105 cells/well, respectively. After 1 day, the cells were treated with SAP, SP, or Pheo for 9 h in the dark and irradiated with a red LED lamp (0.9 J/cm2). After 16 h from light activation, cells were washed with PBS, trypsinized, and pelleted. Pellets were suspended in 100 μL Hepes buffer added with 2 μL of annexin V and 2 μL of propidium iodide, PI (Annexin-V FLUOS Staining kit, Roche, Penzberg, Germany) and incubated for 15 min at 25 °C in the dark. After the addition of 400 μL of buffer, the cells were immediately analyzed by FACScalibur (Becton-Dickinson, San Jose, United States). A minimum of 104 cells per sample was acquired in list mode and analyzed by FlowJo software. The cell population was analyzed by

φΔsystem = φΔPheoπ system/π Pheo The photodegradation rate was determined from the plot of the decrease in the absorbance intensity (ΔA667 nm) versus time. Pheo, SP and SAP (V = 2 mL, [Pheo] = 70 μM) were irradiated, within a 1 cm cuvette, with the same red-LED apparatus for 90 min (fluence ∼ 14.0 J/cm2) and acquiring UV/vis spectra every 10 min. Cell Culture Experiments. Two different cell lines were used: human breast carcinoma MCF-7 and human prostate carcinoma PC3. MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), while PC3 cells in RPMI. Both media contained 10% FBS, antibiotics (penicillin 100U/mL, streptomycin 100 μg/mL), and 2 mM glutamine. All the components were from Euroclone, Milan, Italy. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2 air. The experiments were performed using cells at the exponential growth phase. Nanoassemblies were prepared in PBS as above-described (see Materials). Nanoassemblies labeled with dansyl group (Dns) were obtained as reported in the Supporting Information. Control of free Pheo was prepared in dimethyl sulfoxide (DMSO) and dispersed in PBS. Cells were treated with free Pheo, SP or SAP, or free Pheo, Dns-SP or Dns-SAP (at equal concentration of Pheo), respectively. The volumes of nanoassemblies dispersion (with or without Dns-) were established considering Pheo loading. After the preparation of the assemblies, the formation and the amount of the complexes were checked by UV/vis spectra with a JASCO spectrophotometer, by using correspondent calibration curves (see Figure S4). Fluorimetric Determination of Cellular Uptake. MCF-7 and PC3 cells were seeded in a 24-well plate at a cell density of 5 × 104 cells/well. After 24 h, the cells were treated with Pheo, SAP, and SP at a concentration of entrapped Pheo of 1 μM. At different times of dark incubation (1, 3, and 24 h), the cells were harvested, washed twice, resuspended in 150 μL of PBS, and analyzed by florescence activated cell sorting (FACS) using a FACSARIA 3 (Becton Dickinson, San Josè, USA) equipped with 633 nm laser. A minimum of 10 000 cells per sample was acquired in list mode and analyzed using FLOWJO software. The signal was detected by APC-A in a log scale. Results were normalized to the inherent fluorescence in the untreated control. Competition Assay. Competition experiments were carried out incubating the cells for 1 h with 1 mM free FA (Sigma, Aldrich) prior to the addition of SAP or SP in order to saturate FR-α present on the cell surface. The conjugates were left on cells for 2 h before washing twice in PBS to remove any inbound complexes. Cell uptake was determined by measuring the fluorescence intensity of Pheo using a FACSARIA as reported above. In these experiments both cells were cultured in FA-deficient RPMI medium (Sigma, Milan, Italy). We also compared the uptake of conjugates in different RPMI medium 2533


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Figure 1. Magnification of the 2D-ROESY spectrum of S/Ada-FA in D2O at r.t. (a); model of the interactions between S and Ada-FA, as deduced by ROESY (b).

Table 1. Overall Properties of SP and SAPa System SP

SAP

Dispersion medium H2O PBS pH 7.4 NaCl (0.9% wt) H2O PBS pH 7.4 NaCl (0.9% wt)

DH (nm ± SD) (%)b,c 351 364 357 352 358 368

± ± ± ± ± ±

40 55 66 39 39 56

(100) (100) (100) (100) (100) (100)

PDI

ζ (mV ± SD)

≤0.23 ≤0.30 ≤0.30 ≤0.22 ≤0.22 ≤0.30

−40.0 ± 5.0 − − −51.0 ± 11.2 − −

Theoretical loading (%)d,e 6.62

e

6.12e 7.58d

Actual loading (%)f,g

EE (%)h,i

5.26 ± 0.11

79 ± 1.7

g

5.24 ± 0.09g 7.50 ± 0.05f

86i ± 1.5 99h ± 0.7

a

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, and ζ-potential in ultrapure water. SD was calculated on three different batches. bSize with corresponding intensity % distribution. cSize with corresponding number % distribution. dTheoretical loading of Ada-FA. eTheoretical loading of PS. fActual loading is expressed as the amount of Ada-FA encapsulated in 100 mg of nanoassemblies. gActual loading is expressed as the amount of PS (mg) encapsulated in 100 mg of nanoassemblies. h,icorEntrapment efficiency percentage (EE%): ratio between actual and theoretical loading × 100. h(EE % of AdaFA). i(EE % of PS).

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forward scatter (FSC) and side scatter (SSC) light. The signal was detected by FL1 (annexin V-FITC) and FL-2 (propidium iodide). The dual parameter dot plots combining annexin V-FITC and PI fluorescence show the vial cell population in the lower left quadrant (Q4), the early apoptotic cells in the lower right quadrant (Q3), the late apoptotic (Q2) or necrotic cells (Q1) in the upper right quadrant. Apo-ONE Caspase-3/7 Homogeneous Assay. The Apo-ONE Casapase-3/7 Homogeneous assay (Promega, Milan, Italy) was used to measure the activity of caspase-3 and -7. Following the manufacturer’s instructions, the MCF-7 and PC3 cells were grown in a 96-well plate (density of 7 × 103 cells/well) and exposed to different treatments (SAP, SP, or Pheo). The assay was performed 2 h following light activation. Each well containing 25 μL of sample was added with 25 μL of homogeneous caspase-3/7 reagent, which was diluted 1:100 with buffer. The plate was incubated for 30 min at room temperature before measuring the fluorescence at 521 nm on a fluorescence microplate reader (Spectra Max Gemini XS, Molecular Device) (λexc = 499 nm; λem = 521 nm). In the histogram, we reported the highest value of fluorescence. Statistical Analysis. The data are expressed as means ± standard deviations (SD) of at least 3 experiments. The differences between groups were evaluated with the Student’s t-test and considered significant for p < 0.05.

RESULTS AND DISCUSSION Synthesis of Ada-FA. The synthetic strategy for the preparation of Ada-FA (Scheme 2) has been designed to ensure the interaction of Ada with CD cavities and the

Figure 2. Fluorescence emission spectra and UV/vis (inset) of AdaFA (orange line) in MeOH, SA (black line), and SAP (blue line) in aqueous dispersion (r.t.; λexc = 295 nm). 2534


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Figure 3. Excitation spectra of Ada-FA in MeOH (a), SA (b), and SAP (c) in aqueous solution (r.t.) at different emission wavelengths.

Figure 4. UV/vis (a) and fluorescence emission spectra (b) (λexc = 440 nm) of free Pheo in DCM (green line), SP (red line), and SAP (blue line) in PBS solution, pH 7.4 at r.t.. In all the dispersions Pheo concentration was 7 μM.

Figure 5. Time-resolved fluorescence decays (a) and time-resolved fluorescence anisotropy (b) of Pheo (green circles) in DCM, SP (red circles), and SAP (blue circles) in PBS (10 mM, pH 7.4) at r.t. In all the dispersions Pheo concentration was 7 μM (λex = 390 nm, λem = 675 nm).

through its γ-carboxyl moiety to maintain a high affinity for the FR-α receptor. We chose to link the FA and Ada units to the distal ends of a 2,2′-(ethane-1,2 diylbis(oxy))diethanamine linker by two subsequent coupling reactions; the first between tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (1) and adamantyl carboxylic acid (2) and the second with FA (5, Scheme 2). Matrix assisted laser desorption/ionization time of flight (MALDI-TOF) analysis unambiguously confirmed the expected mass values for the Ada-FA conjugate (6, Scheme 2), exhibiting the values of protonated and sodiated species at 734.02 m/z (MH+) and 755.95 m/z (MNa+), respectively. The almost exclusive γ-conjugation was assessed by mono- and bidimensional NMR experiments (1H and 13C spectra; COSY,

Table 2. Fluorescence Emission Properties of Pheo, SP, and SAP System a

Pheo SPb SAPb

τ1 (ns)c

τ1A1 (%)c

τR (ns)

5.6 6.2 6.2

100 100 100

0.6 ≥20 ≥20

a

In DCM. bIn PBS (10 mM, pH = 7.4, r.t.). cFluorescence lifetimes were measured at λexc = 390 nm and λem = 675 nm; Ai is the amplitude of the intensity decay.

recognition of FA by the FR-α. Accordingly, two main elements have been considered: (1) the linker length should be sufficient to guarantee the complexation of Ada with CD exposing FA residue; (2) FA should be covalently linked 2535


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Figure 6. (a) Micro PL map of the squared area marked in panel a performed using an incident beam at λexc = 470 nm at normal incidence in reflection mode. (b) AFM image acquired in semicontact mode. (c) Line profile of SAP taken along the green horizontal line marked in panel b; (d) PL spectrum of SAP shown in panel a. The background emission shown in the spectrum is due to the glass substrate on which the sample was deposited.

(H12/H15), and 6.73 (H13/H16) ppm unambiguously indicated the complex formation. The adamantane moiety was expected to be included in the CD cavity due to the high binding affinity (Ka on the order of 104−105 M−1).72,73 However, the interaction of FA unit with CD cavity cannot be excluded considering the ability of FA to thread into the β-CD ring74 and the excess of CD cavities in the S/Ada-FA complex ([S] = 2 mM, [Ada-FA] = 1.1 mM, at 1.8/1 molar ratio). Literature data74 indicated that the aminobenzoic unit of FA is projected into the middle of the CD cavity; the pteridine ring adopts a position at secondary hydroxyl groups while the glutamate residue prefers the position at primary hydroxyl groups. The low concentration of Ada-FA in S/Ada-FA prevented the clear detection of complex geometry by ROESY experiments, thus the system was stressed by increasing Ada-FA concentration ([S] = 2 mM, [Ada-FA] = 1.8 mM in D2O at 1/0.9 molar ratio). In the ROESY spectrum, cross peaks between H-3, H-5 protons of CD cavity (3.57 ppm) and peaks at 2.12 (H-22 of the glutamate segment), 1.72 (Ada protons), 0.78 (CH3 thioalkyl chain of CD) ppm confirmed the inclusion of Ada moiety into CD cavity and suggested that the glutamate residue of FA is also in a hydrophobic environment (i.e., a second cavity, not adjacent to the first one, but belonging to a close CD layer, see Figure 1). The strict interaction between two amphiphilic CD units was supported by the detected correlation between cavity and thioalkyl chains, according to the model depicted in Figure 1.

Figure 7. Stability of SAP in aqueous solution, PBS pH 7.4, NaCl 0.9 wt % and PBS in the presence of 2% v/v human serum albumin (HSA) and fetal bovine serum (FBS). In all dispersions Pheo concentration was 7 μM, r.t.

HSQC, and HMBC; see discussion in the Supporting Information). Interaction Study of Ada-FA with S. The successful preparation of the S/Ada-FA complex was confirmed by NMR spectroscopy. The Ada-FA conjugate was nearly insoluble in D2O, but some characteristic peaks were clearly identified in the 1H NMR spectrum of the complex in D2O. In particular, the resonances in the aromatic region at 9.20 (H7), 7.54

Figure 8. Time-dependent FACS uptake of SAP and SP in MCF-7 (a) and PC3 cells (b). Both cells were seeded at a density of 5 × 104 cells/well in a 24-well plate. After 24 h, the cells were incubated with SAP or SP ([Pheo] = 1 μM) in the dark for 1, 3, and 24 h before measuring the mean fluorescence of Pheo. 2536


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DLS analysis of SP and SAP shows a size distribution centered at a hydrodynamic diameter (DH) of about 350 nm and ζpotential of about −40 and −50 mV, respectively (see also Figure S1). These data suggest the presence of more monodispersed drug-loaded assemblies vs amphiphilic CD precursor free or entrapping Ada-FA (see Table 1). Unlike DLS, which generally detects hydrodynamic sizes of the aggregates, transmission electron microscopy (TEM) can reveal dehydrated smaller particles or particle clusters. Indeed, TEM indicated the presence of assemblies clusters ranging between 200 and 300 nm size composed of even smaller aCD nanoparticles (Figure S2). Nanoassemblies possess a charge surface which can change dependently of exposed species to the solvent. In particular, the more negative ζ-potential of SA and SAP vs SP could be ascribed to the presence of Ada-FA on the external surface of the nanocarriers, in agreement with NMR observations. UV/vis, Steady-State, and Time-Resolved Fluorescence Studies. The interactions of S with Ada-FA (SA) , with Pheo (SP) and with Ada-FA and Pheo (SAP) were investigated by UV/vis, steady-state, and time-resolved fluorescence emission. The interaction of Ada-FA with the cyclodextrin in aqueous dispersion of SA and SAP was investigated at [Ada-FA] = 8 μM. Fluorescence emission and UV/vis spectra of free Ada-FA in MeOH in comparison with SA and SAP are shown in Figure 2. In the absorption region of FA, UV/vis of the systems shows a band centered at 281 nm and a weaker band approximately at 365 nm, plausibly ascribed to amino-phenyl and pterin moieties of folate (see inset of Figure 2).75 By excitation at 295 nm, Ada-FA exhibits a set of emission bands centered at 468 nm, tentatively assigned to pterin, and at 351 and 696 nm to the amino-phenyl portion of FA.76 Both bands are remarkably affected in binary SA and ternary SAP. In particular, the band centered at the shortest wavelength (λmax = 351 nm), which is evident in free FA dissolved in polar solvents too,75 underwent a strong quenching upon assembly with aCD. Similarly, the band at 696 nm was depleted both in binary and ternary nanoassemblies, even if the latter shows typical emission of Pheo at 676 and 720 nm. To assign these bands, excitation spectra were carried out. These spectra (Figure 3) indicated that the band centered at 288 nm in free Ada-FA (in part plausibly relative to the amino-phenyl moiety) remarkably decreased its intensity both in SA and SAP. This was evident by acquiring excitation spectra at 350 (black trace) and 700 nm (green trace). On the other hand, the excitation bands of the pterin group (λem = 468 nm), centered around 370 and 276 nm respectively (red trace), are poorly affected with respect to SA and SAP (the I370/I276 intensity ratio of the excitation bands remained mostly unchanged). Likely, within SA and SAP, the interaction of Ada-FA chains with amphiphilic CD could be driven by a strong interdigitation between FA units leading to intermolecular amino-phenyl stacking and consequently to the fluorescence quenching. On the contrary, the intense emission of FA pterin group mostly suggested a structural model with scarce intermolecular selfstacking of pterins in agreement with ROESY outcomes, thus revealing that the pterin group could protrude out of the CD rim for the recognition of FR-α receptor. Fluorescence time-decays (at λexc = 390 nm) of Ada-FA free and within nanoassembly (Figure S3) are well-fitted by twoexponential profiles that allow to estimate two fluorescence lifetimes (τF see Table S2), the shorter (τ1 ≅ 1.4 ns) and the

Figure 9. Uptake of SAP, SP, and Pheo. (A) FACS results. MCF-7 and PC3 cells were treated in the dark with SAP, SP, and Pheo ([Pheo] = 1 μM). The FACS analyses was performed after 3 h of incubation in the dark. (B) Confocal microscope images. MCF-7 and PC3 cells were seeded on 19 mm coverslips at a density of 3 × 105 and 4 × 105, respectively. After 1 day, the cells were treated with SAP, SP, or Pheo ([Pheo] = 2 μM) for 3 h in the dark in RPMI medium without serum.

Figure 10. Competition uptake experiments. Fluorescence uptake of SAP ([Pheo] = 1 μM) in MCF-7 and PC3 cells in different composition media: RPMI + 10% serum = complete RPMI; RPMI + 10% serum (w/o FA) = RPMI complete without folic acid; RPMI + 0% serum (w/o FA) = RPMI complete without folic acid and serum; RPMI + 0% serum (w/o FA) + 1 mM FA = RPMI complete without folic acid and serum. The cells were pretreated with 1 mM FA then incubated for 2 h with SAP.

Moreover, cross peaks between oligoethylenglycole protons of Ada-FA at 3.21 ppm (H-13′/H-16’) and resonances at 9.03 (H-7), 6.72 (H-12/H-15), 4.23 (H-19), 0.78 (CH3 tioalkyl chain of CD) ppm suggested the self-packing of deeply interdigitated Ada-FA chains and the packing of Ada-FA with the thioalkyl chains of CD. Nanoassemblies Properties. Nanoassemblies were prepared with high entrapment efficiency by hydration of organic film and sonication, following the well- established procedure.36,67 Properties of nanoassemblies are reported in Table 1. 2537


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Figure 11. Metabolic activity (%) of (a) MCF-7 and (b) PC3 cells. The cells were seeded at a density of 7 × 103 cells into a 96-well plate and after 24 h they were incubated with increasing amounts of SAP and SP (at different concentrations of entrapped Pheo, as indicated) for 3 h in the dark and then irradiated with red led light (0.9 J/cm2). A resazurin assay was carried out 24 h after irradiation. Data represent mean values ± SD of three independent experiments. t Test analysis was performed: * p < 0.05, ** p < 0.01.

compounds. The emission features at 674 and 718 nm evident in DCM are red-shifted (Δλ ∼ 2 nm) by interaction with S, and a decrease of their intensity was observed. In order to clarify the interaction of Pheo with S, timeresolved fluorescence and anisotropy measurements were performed. The fluorescence emission decay shows a monoexponential behavior with a lifetime value of 5.6 ± 0.1 ns for free Pheo in DCM (see Figure 5a and Table 2). A slight increase at about 6.2 ns was obtained for both SP and SAP, pointing out the interaction of these fluorophores with the colloidal assemblies. This data indicates that Pheo is reasonably entrapped in monomeric form. The rotational correlation time moves to higher values (>20 ns) both in SP and SAP as compared to Pheo in DCM (see Figure 5b) suggesting the embedding into the nanocarrier. Values so high point out that the PS is rotating together with larger particles. Overall, our results indicated that both with and w/o FA, PS strongly interacts with the aCD assemblies and photophysical studies assessed that it is mostly entrapped in monomeric form within the nanoassembly, thus proposing it a promising candidate for further PDT investigations. Morphology and Microphotoluminescence Studies of SAP on Solid Substrate. In order to investigate the morphological properties joined with optical features of the nanoassemblies, dilute dispersions of SAP were casted on glass and evaporated overnight (Figure 6). Figure 6a shows the Micro PL map with a scan area of 10 μm × 10 μm. Figure 6b shows an in-detail view of the sample morphology in the marked area of Figure 6a. Morphology is characterized by a smooth surface with nanoassemblies randomly distributed on the substrate and characterized by an average lateral size of about 280 nm (see Figure 6c) and fairly in agreement with the TEM investigation. As shown in Figure 6a, the assemblies exhibit a photoluminescence emission and their spectrum (Figure 6d) presents an intense emission band centered approximately at 700 nm, plausibly ascribed to the PS loaded in the colloidal aggregate. Release Studies. Kinetic release profiles of Pheo from SAP in PBS at pH 7.4 and 37 °C were evaluated up to 1 week and compared with the stability profile of free Pheo in PBS at the same conditions (Figure S5). Released Pheo was determined exploiting the calibration curve by fluorescence emission of Pheo (Figure S6). No initial burst release was observed from SAP, together with a very slow release, leading to a final ∼6%

Table 3. IC50 Values of SAP and SP in MCF-7 and PC3 Cells IC50 values (nM) SAP SP

MCF-7 cells

PC3 cells

270 600

700 >1000

longer one (τ2 ≅ 7.5 ns for Ada-FA), the latter was previously reported for FA.76 Both values became longer in SA (τ1 ≅ 3.9 ns and τ2 ≅ 8.8 ns), by nearly reversing the distribution amplitudes vs lifetimes of the free conjugates. These increased values were likely due to the presence of a colloidal environment, thus confirming the entanglement of Ada-FA in the amphiphilic carrier. Unfortunately, it was not possible to determine τ values of Ada-FA within SAP, because of the overlapping of the Pheo B-band around 414 nm and the FA absorption band which has a much lower extinction vs the PS B-band. UV/vis revealed that both S and SA interact with Pheo, influencing its spectrum. Figure 4a shows that the absorption spectrum of free Pheo in DCM displays a B-band centered at 414 nm and four Q-bands at 508, 538, 608, and 667 nm. The latter is of relativity high intensity and representative of the chromophore in its monomeric form. Upon complexation with aCD, a slight red-shift (≈4 nm) together with the appearance of a shoulder around 686 nm were observed in the absorption spectra of the nanoassemblies dispersed in PBS (see Figure 4a). The presence of two components might be ascribed to the probable onset of a PS monomer−dimer equilibrium. In fact, it is known that Pheo undergoes dimerization in aqueous solution, and PS aggregates may also be formed on increasing dye concentration.77 However, Pheo dimers are not emissive71 and hence the presence of these self-oligomers should not affect the photodynamic properties of the assemblies. Moreover, the band centered at 686 nm shows hypochromicity and broadening with respect to free Pheo, as observed by a decrease of the extinction coefficient. The values of extinction coefficients of Pheo, εPheo(DCM), Ada-FA, εAda‑FA(MeOH) and of the two nanoassemblies in PBS solution, εSP(PBS) and εSAP(PBS) were determined using Beer’s law (see Figure S4). Steady-state fluorescence spectra of free Pheo (DCM) and PBS dispersions of SP and SAP are reported in Figure 4b. All the spectra show two banded patterns which are typical of chlorophyll-type 2538


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Figure 12. Apoptosis studies. (a) Annexin−Propidium iodide assay. The cells were treated with two concentrations (120, 250 nM) of SAP and SP. After 9 h of dark incubation, the cells were light irradiated (red led, 0.9 J/cm2) and 3 h later the Annexin−Propidium iodide assay was performed. (b) Activation of caspase. Caspase 3/7 levels in MCF-7 and PC3 cells treated as reported in the Experimental Section were measured 2 h after light irradiation. The values are the mean ± SD of two independent experiments expressed as T/C × 100, where T is fluorescence of treated cells and C the fluorescence of untreated cells.

dispersion in NaCl and PBS at pH 7.4 which displays a broad band with a maximum at 683 nm, probably due to the overlapping absorption of the two species. Only slight changes into the bands pattern were observed in ultrapure water, aqueous solution of NaCl 0.9% (w/w) and PBS at pH 7.4 (Figure S7a), while for the solution containing serum proteins (Figure S7b), an enhancement of a nonzero absorption coefficient was detected likely due to the turbidity of the dispersions. The DH values of nanoassemblies dispersions were monitored for up to 2 weeks. At t = 0, SAP exhibits a size distribution with a mean diameter roughly of 350 nm (Figure 7). After 2 weeks, the size of nanoassemblies significantly increases in HSA and FBS (>1 μm). However, only a moderate increase (≤100 nm) was observed for the other dispersions. Interestingly after 24 h, which is reasonably close to the average time spent by nanoassemblies to reach the tumor, the average size of SAP in PBS/HSA or PBS/FBS increases to a maximum of ∼30% vs t = 0. This observation could assess a fair propensity of SAP to repel serum proteins due to the oligoethyleneglycole fringe. Also, the low tendency to disassemble in biologically related conditions can be crucial in the modulation of intratumoral drug release, in agreement with our previous reports.37 To get insight on nanoassembly charge surface, the ζpotential values vs time were measured in ultrapure water (see Figure S8). A strong change of surface charge of SA (ζ ≅ −48

of released Pheo in external medium. The unreleased amount of Pheo was 64.3 μM, which is ∼80%, whereas an amount of PS remains attached on the dyalisis membrane as Pheo selfoligomers (corresponding to 14%, data not shown). Actually, in the same experimental conditions, the precipitation profile of free Pheo as a function of time was monitored (inset in Figure S5), showing that ∼77% of initial amount of Pheo was still dispersed in solution within 1 week, whereas ∼23% precipitated or was linked to the membrane. In agreement with these results, it is possible to admit that after 1 week, a measured amount of Pheo can be released from a nanoassembly (∼20%) and distributed between the release medium and the membrane. Almost the same percentage of free PS (previously dissolved in DMSO/H2O) precipitated in PBS, pointing out the indispensable role of aCD nanoassemblies in dispersing the PS and sustaining its release for 1 week. Stability of Nanoassemblies. In order to investigate if the aggregation properties of SAP are influenced by a dispersing medium, we carried out stability studies vs time in water and in biologically relevant media by DLS and UV/vis. The absorption spectra were recorded at two different times (t = 0; t = 1 week) (Figure S7). For freshly prepared SAP at t = 0, all the absorption spectra show two bands related to the equilibrium monomer−dimer with different ratios of A671/A695. In particular, the equilibrium appears shifted toward the monomeric form for all the nanoassemblies, except for the 2539


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Biomacromolecules mV) vs unloaded S (ζ ≅ −36 mV) and SAP (ζ ≅ −50 mV) vs SP (ζ ≅ −40 mV) (see Table 1) suggests that FA with its additive negative charge (due to the free α-carboxylic group) is plausibly placed on the nanoassembly surface. In all the investigated dispersions of PS-loaded nanoassemblies, highly negatives value of ζ-potential were observed, indicating a good stability of these nanoassemblies in water. It is worth noting that these dispersions are fairly stable even for 2 weeks after their preparation. Comparative Singlet-Oxygen Generation Measurements. The photodynamic potential of SP and SAP was explored by typical indirect detection of 1O2 generation. For this experiment, two samples with a concentration of ∼70 μM for both systems were used to produce a detectable 1O2 amount. In our experimental conditions, photobleaching of free Pheo and Pheo-loaded aCD along the irradiation time can be ruled out, as we observed photostability higher than 95% for both free and entrapped Pheo (data not shown). The investigated samples generate appreciable amounts of 1O2 SAP (φSP = 0.39) in comparison with free Pheo Δ = 0.62), (φΔ Pheo (φΔ = 0.59) (Figure S9). Pheo in the SP shows a similar singlet oxygen quantum yield with respect to the free one, according to lifetime values that indicate the monomeric nature of the fluorophore. On the contrary, Pheo in the ternary system displays a decrease of singlet oxygen quantum yield. This fact could be ascribed to the oxygen quenching of the pterin moiety by the folate group, which could act as a singlet oxygen trap.78 Uptake of SAP and SP in MCF-7 and PC3 Cancer Cells. The final goal of the present work was to demonstrate that FA-decorated aCD nanoassemblies deliver Pheo via a FRα receptor, to achieve PDT action with improved selectivity. FR-α is overexpressed on the surface of breast, ovarian, cervical, and colorectal cancer cells as well as relatively absent in normal tissues.79 To test SAP and SP, we carried out the biological experiments in two human tumor cell lines with different expressions of the FR-α receptor: breast cancer cells (MCF-7) and human prostate cancer cells (PC3). The first is known to express a high level of folate receptor FR-α.80−82 The second shows a low level of FR-α expression (Figure S10)83 as well as a lack of the prostate specific membrane antigen (PSMA).84−87 In order to study the receptor-mediated uptake and to follow the cell internalization of nanoassemblies, we performed a time-dependent uptake experiment by flow cytometry. Figure 8a,b reports the mean fluorescence values obtained with MCF-7 and PC3 cells incubated for 1, 3, and 24 h in the dark with SAP or SP. SAP was internalized into the cells more efficiently than SP. MCF-7 cells showed a more marked difference between the two nanoassemblies than PC3 cells. The best difference in uptake between the two nanoassemblies was observed after 3 h of incubation: the ratio of SAP/SP was 2 in MCF-7 cells and 1.2 in PC3 cells. We therefore decided to analyze the uptake of SAP and SP by confocal microscopy at this time. We used Hoechst to stain the nuclei of the cells and followed the uptake of the nanoassemblies by measuring the red fluorescence between 580 and 778 nm emitted by Pheo upon excitation at 570 nm. Figure 9 shows that in MCF-7 cells, which express a high level of FA receptor, the uptake of SAP is higher than SP. In contrast, in PC3 cells, which have a lower level of FA receptor, SAP and SP show similar uptake, as expected. We also report images about the uptake of free Pheo in both cell lines. The images show that the uptake of free Pheo is higher

to that of SAP. Instead, the FACS data of Figure 9A show that free Pheo is taken up by the cells more than Pheo incorporated into the nanossemblies. However, we expect that in vivo, Pheoloaded nanoassemblies should be more tumor specific than free Pheo, due to the enhanced permeability and retention effect88 and sustain the release of photosensitizer in the tumoral tissue. It is also evident from the confocal images (Figure 9b) that both SAP and SP are localized in the cytoplasm of the cells (see also Figure S11) With the aim to follow the cell uptake and localization of both amphiphilic CD and PS of SAP, we labeled S with an amphiphilic cyclodextrin modified with Dns groups (SC6Dns) forming dansylated aCD nanoassemblies.89 In order to monitor the PS-loaded nanoassembly, with Ada-FA (SAP) or without it (SP), Dns-SP and Dns-SAP were formulated as a double fluorescent nanoassembly in which Pheo emits in the red region and Dns group in the green, respectively. Indeed, as an example, the fluorescence emission spectrum of Dns-SP exhibits both a double patterned emission band of Pheo centered at 676 and 715 nm, and the typical emission band centered at ≅484 nm ascribed to the Dns moiety (Figure S12). Also, Dns-SAP was obtained and checked by confocal microscopy, whereas the fluorescence emission band of Dns was completely overlapped with FA band (data not shown). The confocal images (Figure S13) confirm that Dns-SAP enters more efficiently into MCF-7 with respect to the PC3 cells and that the amphiphilic CD carrier can colocalize with Pheo in the cytoplasm of the cells. To support the uptake further, we performed competition experiments. MCF-7 and PC3 cells were treated with 1 mM free FA in order to saturate the folate receptor (Figure 10). It can be seen that when FA is removed from the medium, the uptake of SAP increases by 33% only in MCF7 cells. As expected, when we used a medium without both FA and 10% serum, the uptake increased by a further 8%. But, when we added 1 mM FA, we observed a decrease in the uptake, due to the competition effect of the folic acid. These effects were observed in a much lower extent in PC3 cells, as they scarcely express the FA receptor. The results confirmed the role of the FA for SAP uptake. Phototoxicity of SAP and SP in MCF-7 and PC3 Cells. The phototoxicity of FA-decorated nanoassemblies was evaluated. First, we confirmed that SAP and SP are not toxic in the dark (Figure S14c). The MCF-7 and PC3 cells were treated with increasing doses of the nanoassemblies, incubated in the dark for 3 h and subsequently irradiated with a red LED light (0.9 J/cm2). After 24 h, cell proliferation was determined by resazurin assay. Figure 11 shows the plots obtained from dose−response curves. In MCF-7 cells (Figure 11a), SAP showed a significant decrease of cell viability in comparison to SP in the range 250−700 nM (p < 0.05 at 250 nM dose; p < 0.01 between 500 and 700 nM doses). In PC3 cells (Figure 11b), the difference between SAP and SP was not strong, even if SAP showed a better effect than SP at a dose of 500 nM (p = 0.05). Excluding the influence on phototoxicity of S, FA, and their binary SA assembly (see Figure S15), we found that there was a major photodynamic effect of SAP in MCF-7 with respect to PC3 cells (Table 3). SAP turned out to be more potent than SP in both MCF-7 and PC3 cells (IC50 = 270 nM vs IC50 = 600 nM in MFC-7 and IC50 = 700 nM vs IC50 = >1000 nM in PC3), indicating an efficient internalization by the FA receptors. We also found that both SAP and SP showed a lower activity than free Pheo in both types of cells (Pheo IC50 2540


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values were 180 and 280 nM in MCF-7 and PC3, respectively) (Figure S14). Free Pheo was prepared by dissolution it in DMSO and dilution in aqueous solution, whereas Pheo formulated in SAP benefits with respect to free Pheo of an higher dispersibility in biologically relevant media. Furthermore, FACS experiments ascertained the level of ROS photogenerated within the cells pointing out to a dosedependent increase of ROS induction by SAP, SP and free Pheo (see Figure S16). In order to evaluate the cell death mechanism, we carried out apoptotic assays. MCF-7 and PC3 cells were treated with different amounts of SAP and SP, and 16 h after light irradiation an Annexin−Propidium iodide assay was performed. Figure 12a shows an increase of cell population in Q2 (Annexin and PI both positive) due to late apoptosis. The Q2 population in MCF-7 cells treated with SAP was nearly double than in the MCF-7 cells treated with SP. In contrast, in PC3 cells, the Q2 population was similar in both treatments. To further support apoptosis, we carried out a caspase 3/7 assay, 2 h after light irradiation (Figure 12b,c). The caspases increased in a dose−response manner in both types of cells treated with SAP and SP. This was observed also in the cells treated with free Pheo. Altogether, the data suggested that apoptosis is the first route of cell death promoted by SAP and SP after light irradiation. Finally, these results, in agreement with previous outcomes in which nanocarriers entrapping Pheo or other PSs44,48 showed improved biodistribution and high tumor uptake, pointed out that the proper control of molecular components assembly, its physicochemical features and the preliminary investigation of death cell mechanisms, should be considered a preparatory strategy to get multifaceted nanosystems, such as SAP, for successful in vivo targeted PDT.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00306. Material and Methods: synthesis and characterization of Ada-FA (synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)adamantane-1-carboxamide, synthesis of Ada-FA); synthetic strategy for the preparation of Ada-FA (Scheme S1); trasmission electron microscopy, time resolved fluorescence; release studies; Western blot analysis; intracellular ROS detection; double-fluorescent nanoassemblies preparation; confocal microscopy studies on double fluorescent nanoassemblies; confocal microscopy studies on double fluorescent nanoassemblies. Figures: size distribution and ζ-potential of SP and SAP in ultrapure water (Figure S1); properties of S and SA (Table S1); trasmission electron microscopy images (Figure S2); time resolved fluorescence decay of Ada-FA and of SA (Figure S3); fluorescence emission lifetimes of Ada-FA and SA (Table S2); absorbance calibration curves of Pheo, Ada-FA, and SP and SAP (Figure S4); release kinetics of Pheo from SAP (Figure S5); calibration curve by fluorescence emission for Pheo in PBS (Figure S6); stability studies in biological relevant media by UV/vis spectroscopy (Figure S7); stability of SP and SAP in aqueous dispersion by ζ-potential measurements (Figure S8); RNO bleaching kinetics of Pheo, SP, and SAP (Figure S9); different expression of folate receptor protein (FR-α) by Western blotting (Figure S10); confocal microscope images of cells treated with Pheo, SP, and SAP (Figure S11); fluorescence emission spectra of Dns-SP in aqueous solution (Figure S12); confocal microscope images of cells treated with Pheo, Dns-SP, and Dns-SAP (Figure S13); metabolic activity (%) of cells treated with Pheo, SP, and SAP in PC3 and MCF-7 cells (Figure S14); metabolic activity (%) of S, Ada-FA, and SA in MCF-7 and PC3 cells (Figure S15); intracellular ROS of cells treated with Pheo, SP, and SAP (Figure S16) (PDF)

CONCLUSIONS

The design of a nanoassembly based on aCD decorated with active targeting groups was here proposed by proper finetuning of the chemico-physical features of the components. Amphiphilic cyclodextrins, bearing a suitable PEG (1−2 kDa), able to minimize undesirable interaction with serum protein, tailored with folic acid and entrapping pheophorbide-A as a photosensitizer were designed as FR α-cell targeting nanophotherapeutics. The Ada-FA conjugate was incorporated by means of a strong interaction between the Ada group and the aCD cavity. Spectroscopic data agree that the FA moiety can protrude on the external surface from the outer aCD rim, thus allowing a selective delivery (within 24 h) of PS within cancer cell expressing high level of FR-α vs cells with a low level of FR-α. These actively targeted nanoassembies are stable in aqueous solution and in biologically relevant media until 2 weeks, showing a low tendency to disassemble and to increase their size in the presence of serum protein. FA-decorated nanoassemblies have good propensity to generate 1O2, sustain release of PS within 1 week, and show improved phototoxicity in cancer cells FR-α(+) vs not decorated ones. Indeed, SAP turned out to be more potent than SP in both MCF-7 and PC3 cells indicating an efficient internalization mediated by the FA receptors. More importantly, these nanophotherapeutics have a high PDT activity within FR-α(+) vs FR-α(−) cells, selecting the target site dependently from FR amount expression.

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AUTHOR INFORMATION

Corresponding Author

*Antonino Mazzaglia. Email: [email protected]. ORCID

Anna Piperno: 0000-0001-6004-5196 Angela Scala: 0000-0003-2171-9033 Luigi E. Xodo: 0000-0003-3344-7207 Luigi Monsù Scolaro: 0000-0002-9742-9190 Antonino Mazzaglia: 0000-0002-3140-5655 Author Contributions ⊥

R.Z., V.R. and A.P. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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. We are grateful to Prof. Placido Mineo for Maldi and to Dr. Violetta Borelli (Department of Life Sciences, University 2541


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of Trieste) for providing LED apparatus. We thank Dr. Francesca D’Este (Department of Medical Area, University of Udine) for performing the confocal images.

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