Topology effects in photodynamic therapy with phthalocyanine nanocarriers

PAPER Magali Gary-Bobo, José A. Pomposo, Fabienne Dumoulin et al . Topology effects in photodynamic therapy with phthalocyanine nanocarriers Materials Advances rsc.li/materials-advances ISSN 2633-5409 Volume 6 Number 1 7 January 2025 Pages 1–466 148 | Mater. Adv., 2025, 6, 148–156 © 2025 The Author(s). Published by the Royal Society of Chemistry Cite this: Mater. Adv., 2025, 6, 148 Topology effects in photodynamic therapy with phthalocyanine nanocarriers† Davide Arena, a U¨mit I˙s-ci, b Me ´lanie Onofre, c Christophe Nguyen, c Zeynel S-ahin, b Ester Verde-Sesto, ad Amaia Iturrospe, a Arantxa Arbe, a Magali Gary-Bobo, * c Jose ´A. Pomposo * ade and Fabienne Dumoulin * f Two different amphiphilic copolymers, a random copolymer (RCP) and a block copolymer (BCP), have been used to encapsulate a far-red photosensitizing hydrophobic zinc phthalocyanine while forming self-assembled nanocarriers of different topology: self-folded single-chain nanoparticles (SCNPs) and star-like aggregates (SLAs), respectively. Different copolymer/phthalocyanine ratios have been tested to assess their effect on structural properties which were determined by small-angle X-ray scattering (SAXS) measurements. The relevance of these materials as photosensitizer nanocarriers for photodynamic therapy (PDT) has been studied against human breast cancer cells (MCF-7). A better PDT effect was found for the SCNP-Pc2 x (100 mgmL 1 ) excited at 800 nm with a pulsed laser than for the SLA-Pc2 x under identical conditions. Conversely, irradiation at slightly lower wavelengths (740 nm) of MCF-7 cells incubated with SLA-Pc2 x resulted in a notorious PDT effect when compared to that observed for MCF-7 cells incubated with the SCNP-Pc2 x . These systems represent new strategies for the encapsulation of photosensitizers for photodynamic therapy. 1. Introduction Photodynamic therapy (PDT) is now a well-recognized treatment of cancer, even though it is still too often described as emerging. The first photosensitizers were approved decades ago, 1,2 but had several drawbacks in terms of characterization, tumor targeting and efficiency. Second generation-photosensitizers have since been approved or are in advanced clinical trials. 3–5 Using excitation wavelengths belonging to the first phototherapeutic window (600–1000 nm) for PDT is advantageous for two reasons: first, it avoids exciting endogenous chromophores, 6 and second, it allows deeper light penetration into biological tissues than with shorter wavelengths. 7 Phthalocyanines are synthetic tetrapyrrolic molecules of the porphyrinoid family and show typically an average maximum absorption at 700 nm. In this respect, phthalocyanines are excellent photosensitizer molecular bases for anticancer PDT. 8–11 Many water-soluble phthalocyanines with cationic, anionic or neutral substituents have been used for PDT. 12 However, they are frequently aggregated, which impairs their efficiency. Besides, excessive water-solubility of photosensitizers may be detrimental since it induces a rapid clearance from the body without having the chance to accumulate in the targeted tissues, and/or prevents cellular internalization by limiting the cellular membrane crossing. The use of hydrophobic photosensitizers overcomes these issues, but a significant drawback is that they are not biocompatible as they cannot circulate in the bloodstream. This promoted the use of various nanocarriers, either by encapsulation or by covalent grafting of the hydrophobic photosensitizer. 13 An additional advantage is that nanoformulated photosensitizers can benefit from the enhanced permeability and retention (EPR) effect, which may have some limitations 14 butisanywayagoodwaytodeliverphotosensitizers. A liposomal formulation of unsubstituted zinc phthalocyanine has entered advanced clinical trials, 15 and several micellar formulations of phthalocyanine derivatives for PDT applications have been reported. 16,17 Conjugating a mono-hydroxylated phthalocyanine to poly-L-glutamic acid allowed to obtain polymeric nanoparticles of high efficiency, 18,19 and phthalocyanine-based organo-silica nanoparticles exhibited remarkable photodynamic a Centro de Fı ´sica de Materiales (CSIC, UPV/EHU) and Materials Physics Center MPC, Paseo Manuel Lardizabal 5, 20018 Donostia, Spain. E-mail: josetxo[email protected] b Marmara University, Faculty of Technology, Department of Metallurgical and Materials Engineering, 34854 Maltepe, Istanbul, Tu ¨rkiye c Institut des Biomole ´cules Max Mousseron, CNRS, ENSCM, 34293 Montpellier, France. E-mail: [email protected]r d IKERBASQUE–Basque Foundation for Science, Plaza de Euskadi 5, 48009 Bilbao, Spain e Departamento de Polı ´meros y Materiales Avanzados, Fı ´sica, Quı ´mica y Tecnologı ´a, University of the Basque Country (UPV/EHU), 20800 Donostia, Spain f Acıbadem Mehmet Ali Aydınlar University, Faculty of Engineering and Natural Sciences, Department of Biomedical Engineering, 34752 Atas-ehir, Istanbul, Tu ¨rkiye. E-mail: [email protected] †Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d4ma00524d Received 21st May 2024, Accepted 5th November 2024 DOI: 10.1039/d4ma00524d rsc.li/materials-advances Materials Advances PAPER Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 148–156 | 149 efficiency. 20,21 It has been shown that the nature of the polymeric nanocarrier can greatly affect the photodynamic efficiency of a nano-photosensitizing system. 22 Self-assembled nanocarriers such as single-chain nanoparticles (SCNPs) have proven to be efficient carriers for the delivery of hydrophobic drugs. 23–25 These works are relatively recent. Indeed, only a handful of tetrapyrrolic derivatives combined with SCNPs have been reported: a porphyrin has been grafted onto the skeleton of individual SCNPs; 26 phthalonitriles (which are the most common precursor of phthalocyanines) have been introduced on a polystyrene backbone, the subsequent intramolecular formation of the phthalocyanine moiety leading to the formation of colored single-chain polymeric nanoparticles; 27 and the axial coordination of a polymeric chain containing pyridine moieties by a cobalt phthalocyanine produced CO 2 -reducing materials. 28 In this work, a photosensitizing far-red absorbing hydrophobic zinc phthalocyanine has been designed to be encapsulated into two different polymeric nanocarriers prepared from the same monomers. The random copolymer self-assembled into selfassembled single-chain nanoparticles (SCNPs), and the block copolymer formed star-like aggregates (SLAs). A careful characterization of the resulting phthalocyanine-loaded nanocarriers (SCNP-Pc2 x and SLA-Pc2 x ) was carried out by means of smallangle X-ray scattering (SAXS) experiments. Finally, we report how the very different topology of these two nanocarriers modulate the PDT efficiency on MCF-7 breast cancer cells. 2. Experimental 2.1. Chemistry 2.1.1. Materials and methods. Oligo(ethyleneglycol)monomethylether methacrylate (OEGMA 300 ) (99%), (2-acetoacetoxy)- ethyl methacrylate (AEMA) (95%), 2,20-azobis(2-methylpropionitrile) (AIBN) (Z98%), 1,4-dioxane, n-hexane, deuterated chloroform (CDCl 3 ) (99.96 atom% D, containing 0.03% (v/v) tetramethylsilane, TMS), sodium phosphate dibasic heptahydrate (Na 2 HPO 4  7H 2 O), sodium phosphate monobasic monohydrate (NaH 2 PO 4  H 2 O) were purchased from Sigma-Aldrich and used, unless specified, as received. 4-Cyanopentanoic acid dithiobenzoate (CPADB) (Z97%) was purchased from Strem Chemicals. Methanol and tetrahydrofuran (THF) (HPLC grade) were purchased from Scharlab. AIBN was recrystallized from methanol. OEGMA 300 and AEMA were purified by passing through basic alumina. 3-Nitrophthalonitrile, isobutylthiol, hexadeuterated dimethylsulfoxide (DMSO-d 6 ), dimethylformamide (DMF), potassium carbonate (K 2 CO 3 ), dimethyl sulfoxide (DMSO), zinc acetate Zn(OAc) 2 , dimethylaminoethanol and dichloromethane were used as purchased. Column chromatographies were done using Merck Silica Gel 60 (0.040–0.063 mm). NMR spectra were recorded at room temperature in CDCl 3 or DMSO-d 6 solutions on a Varian spectrometer (500 MHz for 1 H, 125 MHz for 13 C spectra). FT-IR spectra were recorded between 4000 and 650 cm 1 using a PerkinElmer Spectrum 100 FT-IR spectrometer. Mass spectra were measured on a MALDI (matrix assisted laser desorption ionization) BRUKER Microflex LT (Bremen, Germany) using 2,5-dihydroxybenzoic acid as the matrix. 2.1.2. Synthesis of 3-isobutylthiophthalonitrile (1). 3-Nitrophthalonitrile (5 g, 28.8 mmol), isobutylthiol (2.9 g, 33.2 mmol) and dry potassium carbonate (30 g, 217.5 mmol) were stirred in anhydrous DMF (50 mL) under argon at 40 1C for 24 h until complete disappearance of 3-nitrophthalonitrile in TLC monitoring. Then the reaction mixture was poured into water (500 mL), and the resulting solid was collected by filtration and thoroughly washed with water. The dried crude product was recrystallized from ethanol. Yield: 80% (4.98 g). 1 H NMR (500 MHz, CDCl 3 )d, ppm: 7.61–7.56 (m, 3 H), 2.96 (d, 2H), 1.96 (m, 1 H), 1.11 (d, 6 H). 13 C NMR (125 MHz, CDCl 3 )d, ppm: 146.3, 132.6, 131.1, 129.4, 117.2, 115.3, 114.7, 113.8, 41.9, 28.1, 21.9. FT-IR (n,cm 1 ): 3090, 2973, 2956, 2934, 2230, 1566, 1454, 1421, 1385, 1319, 1259, 1198, 1153, 1083, 958, 792, 726. 2.1.3. Synthesis of phthalocyanine Pc2. A mixture of 1(1 g, 4.62 mmol) and Zn(OAc) 2 (424 mg, 2.31 mmol) was refluxed in dimethylaminoethanol (10 mL) under argon for 12 h. After cooling to room temperature, the reaction mixture was poured into water (100 mL). The resulting precipitate was filtered off and washed several times with ethanol. Pc2 was isolated by chromatography on silica gel using a mixture of dichloromethane/ethanol (100/1) as the eluent. Yield: 28% (300 mg). MALDI-TOF-MS (DHB) m/z: 930.301 [M] +; calculated for C 48 - H 48 N 8 S 4 Zn: 930.588. FT-IR (n,cm 1 ) 3059, 2953, 1592, 1565, 1462, 1430, 1365, 1330, 1311, 1222, 1163, 1103, 1041, 946, 900, 881, 792, 757, 735, 679. 1 H NMR (500 MHz, DMSO-d 6 )d, ppm: 8.97–8.91 (m, 3H), 8.66–8.60 (m, 2 H), 8.06–7.59 (m, 8 H), 3.38– 3.16 (m, 8 H), 2.23 (m, 4 H), 1.31 (m, 24 H). UV-vis (THF): l max , nm (loge): 710 (5.3), 641 (4.6), 340 (4.9). 2.1.4. Synthesis of the random copolymer poly(OEGMA 300 - r-AEMA) (RCP). 10.55 mg (0.038 mmol) of 4-cyanopentanoic acid dithiobenzoate (CPADB) and 1.29 mg (0.0077 mmol) of AIBN, 1.83 mL (6.4 mmol) of OEGMA 300 , 0.31 mL (1.6 mmol) of AEMAa and 3.37 mL of dioxane were added in this order. The system was sealed with a rubber septum and the mixture was then left to degas under inert gas for 15 minutes. After that time, the reaction mixture was maintained at 70 1C, under nitrogen positive pressure and under continuous magnetic stirring for 17 hours. M n (kDa)= 99.6, PDI= 1.04, dn/dc= 0.185. AEMA content (mol%) = 20. 1 H NMR (400 MHz, CDCl 3 ) d, ppm: 4.37 (m, CCO 2  C H  2 CH 2 CO 2 CH 2 ), 4.18–4.11 (m, 2H, CCO 2 CH 2  C H  2 CO 2 ), 3.83–3.58 (m, CH 2 O C H  2 CH 2 ), 3.41 (s, O C H  3 ), 2.33 (s, CH 2 CO C H  3 ), 1.81 (m, CCH 2 ), 1.04 (m, CCH 3 ), 0.87 (m, CCH 3 ). 2.1.5. Synthesis of the block copolymer poly(OEGMA 300 )-bpoly(AEMA) (BCP). 29.25 mg (0.105 mmol) of CPADB and 3.45 mg (0.021 mmol) of AIBN were dissolved in 1.5 mL of anhydrous 1,4-dioxane. Then, 0.6 mL (3.144 mmol) of AEMA was added and the resulting mixture was left degassing purging nitrogen for 20 minutes. After this time, the solution was left stirring under nitrogen positive pressure and at 70 1C for 5 hours. Then, the reaction was quenched by freezing the mixture at 78 1C. 1 mL (0.014 mmol of macro-CTA) of the crude, 6 mL (21 mmol) of OEGMA 300 , 2.3 mg (0.014 mmol) of Paper Materials Advances Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 150 | Mater. Adv., 2025, 6, 148–156 © 2025 The Author(s). Published by the Royal Society of Chemistry AIBN and 18 mL of 1,4-dioxane were degassed purging nitrogen for 20 minutes, then left stirring at 70 1C under positive nitrogen pressure for 17 hours. After this time, the reaction was quenched by cooling the mixture at 78 1C. The polymer was precipitated in a large excess of n-hexane (40) for three consecutive times and dried under dynamic vacuum. M n (kDa) = 71.8, PDI = 1.02, dn/dc = 0.185. AEMA content (mol%) = 14. 1 H NMR (400 MHz, CDCl 3 )d, ppm: 4.37 (m, CCO 2  C H  2 CH 2CO 2 CH 2 ), 4.18–4.10 (m, 2H, CCO 2 CH 2  C H  2 CO 2 ), 3.77–3.57 (m, CH 2 O C H  2 CH 2 ), 3.40 (s, O C H  3 ), 2.32 (s, CH 2 CO C H  3 ), 1.87 (m, CCH 2 ), 1.04 (m, CCH 3 ), 0.87 (m, CCH 3 ). 2.1.6. Characterization of RCP and BCP. Molecular structure and copolymer composition were investigated via 1 H NMR spectroscopy, using a Bruker spectrometer operating at 400 MHz. AEMA content, expressed as %AEMA (mol%) was calculated according to eqn (1): %AEMAðmol%Þ¼ SAEMA COCH3 SAEMA COCH3þSOEGMA OCH3 100 (1) where SAEMA COCH3is the signal intensity of the methylic protons of AEMA functional groups (d: 2.3 ppm) and SOEGMA OCH3is the signal intensity of methoxylic protons of OEGMA moieties (d: 3.4 ppm), both integrated taking SOEGMA OCH3as internal standard. Number-average molecular weight (M n ) and dispersity (Ð) were determined via size exclusion chromatography/multiangle light scattering (SEC/MALS) measurements, which were performed at 30 1C on an Agilent 1200 system equipped with PLgel 5 mm Guard and PLgel 5 mm MIXED-C columns and triple detection: a differential refractive index (dRI) detector (Optilab Rex, Wyatt), a multi-angle laser light scattering (MALS) detector (MiniDawn Treos, Wyatt), and a viscosimetric (VIS) detector (ViscoStar-II, Wyatt). THF was used as eluent at a flow rate of 1 mL min 1 . The topology of RCP and BCP in water was determined from small-angle X-ray scattering (SAXS) measurements on polymer solutions at a fixed polymer concentration of 1 mg mL 1 . SAXS experiments were conducted on a Rigaku 3-pinhole PSAXS-L equipment operating at 45 kV and 0.88 mA. The MicroMax-002+ X-Ray Generator System is composed by a microfocus sealed tube source module and an integrated X-Ray generator unit which produces CuKatransition photons of wavelength l= 1.54 Å. The radius of gyration (R g ) and size scaling exponent (n)ofRCP were determined through fits of the experimental data to a generalized Gaussian coil 29 function. For BCP, analysis of the SAXS data in terms of the Dozier 30 star model provided R g and the average number of arms, f. In both cases the SASfit program was used. 31 UV-vis measurements of RCP and BCP were carried out in water at 25 1C in an Agilent 8453A apparatus with Peltier thermostatic cell holder, T-controller 89090A. 2.1.7. General procedure for the encapsulation of Pc2 into RCP or BCP to give SCNP-Pc2 x and SLA-Pc2 x , respectively. A stock solution of Pc2 (0.25 mg mL 1 ) was prepared by dissolving 1 mg (1.1 mmol) of Pc2 in 4 mL of THF. 10 mg of RCP or BCP were dissolved in 1 mL of THF and Pc2 stock solution was added (186 mL or 447 mL). The resulting solution was left stirring for 48 hours until complete evaporation of the THF and the formation of a thin film. After this time, 10 mL of deionized water was added, and the resulting mixture was left stirring at room temperature for 3 days. Pc2 concentration is therefore either 5 or 12 mM, and the respective nanoparticles are denoted as SCNP-Pc2 x or SLA-Pc2 x (x= 5 or 12). 2.1.8. Characterization of SCNP-Pc2 x and SLA-Pc2 x .SCNPPc2 x and SLA-Pc2 x were characterized by SAXS and UV-vis, following the same procedures used for RCP and BCP, respectively. 2.2. Optical measurements UV-visible electronic absorption spectrum was recorded on a Shimadzu 2001 UV spectrophotometer. Fluorescence quantum yield of Pc2 was determined by the comparative method according to the equation: FF¼FFðstdÞF:AðStdÞ:n2 FðStdÞ:A:n2 ðStdÞ where Fand F Std are the areas under the emission curves for the sample and standard, respectively. Aand A Std are the absorbance at the excitation wavelength for the sample and standard, nis the refractive index of the solvents for the sample and reference. Unsubstituted ZnPc in DMSO was used as a standard (FF (Std) = 0.20) and sample and standard were excitated (at l= 640 nm) at the same wavelength in DMSO. Singlet oxygen quantum yield of Pc2 was obtained in DMSO solution(inair,nooxygenbubbled)byindirectmethodwithzinc phthalocyanines (ZnPc) as a reference and 1,3-diphenylisobenzofuran (DPBF) as a chemical quencher for singlet oxygen, using the fallowing equation: FD¼FStd D RIStd abs RStd Iabs where FStd Dis the singlet oxygen quantum yield for the standard ZnPc (F D =0.67inDMSO).Rand R Std are the DPBF photobleaching rates in the presence of Pc2, and the standard, respectively. I abs and I Std abs are the rates of light absorption by Pc2 and standard, respectively. Mixture of phthalocyanine (B6mM, absorption B1.2 at 717 nm) and DPBF (B30 mM, absorption B0.8 in 417 nm) was irradiated in the Q band region in 10 cycles of 5 s each one. DPBF degradation was monitored at 417 nm and the F D value was determined using the set up described above. 32,33 2.3. Biology 2.3.1. Materials. Human breast cancer cells (MCF-7) were purchased from American Type Culture Collection (ATCC), Dulbecco’s modified Eagle’s Medium (DMEM/F12), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco Life Technology, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar (Thermofisher). 2.3.2. Cell culture. MCF-7 cells were maintained in DMEM/ F12 supplemented with 10% FBS and 1% penicillin/streptomycin. Cells grew at 37 1C, in humidified atmosphere and under 5% CO 2 . Materials Advances Paper Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 148–156 | 151 2.3.3. Cytotoxicity studies. MCF-7 cells were seeded in a plastic multi-well (96) plate. The day after seeding, cells were incubated with increasing doses of nanoparticles (from 0 to 200 mgmL 1 ) for 72 h. The viability of the cells incubated with nanoparticles was evaluated using MTT assay. For this, cells were incubated for 4 h with 0.5 mg mL 1 of MTT in media. The MTT/media solution was then removed, and the precipitated formazan crystals were dissolved in 150 mLofanequalvolume solution of ethanol/DMSO. After 30 min, the optical density (OD) of samples was read at 540 nm using microplate reader. The OD values are directly correlated with the number of living cells in well. Cell viability was calculated as % viability = OD of treated cell/OD of vehicle control 100. 2.3.4. PDT experiments. For continuous excitation (633, 650 or 740 nm), MCF-7 cells were seeded in a plastic multi-well (96) plate. The day after, cells were incubated with 100 mgmL 1 of SCNP-Pc2 x or SLA-Pc2 x for 24 h. After the incubation time, cells were exposed or not to light source at 633, 650 or 740 nm. The excitation at 633 nm (4.96 J cm 2 )wasperformedwithanEVOS 5000 microscope using Cy5 EVOSLEDlightcube during 1 min, 64,9 mW at magnification 4.Theexcitationat650nm (39 J.cm 2 ) was performed with a portable laser during 20 min. The excitation at 740 nm (11.25 J m 2 )wasperformedwithaLED spotlight from EvoluChemtfor 10 min. For pulsed laser excitation (800 nm), MCF-7 cells were incubated in a multi-well (384) plate with glass bottom. The day after, cells were incubated with 100 mgmL 1 of SCNP-Pc2 x or SLA-Pc2 x for 24 h, and then submitted or not to laser irradiation with a confocal Carl Zeiss two-photon microscope at 800 nm and maximum laser power (3 W input, 1100 mW output at lens). The laser beam was focused by a microscope objective lens (CarlZeiss10-foldmagnification/ objective 0.3 EC Plan-Neofluar) and irradiations were performed by 3 scans of 1.5 s each. Two days after irradiation, the phototoxicity effect of nanoparticles was assessed using MTT assay as previously described. Cells treated with the vehicle blank nanocarriers (RCP and BCP) were considered as a control. 2.3.5. Statistical analysis. Student’s t-test was performed to compare paired groups of data. A pvalue o0.05 was considered as statistically significant. 3. Results and discussion 3.1. Molecular design and synthesis of iso-butylthio nonperipherally tetrasubstituted Zn phthalocyanine (Pc2) The photo-properties of phthalocyanines can be modulated by their substitution pattern. 34 The presence of S-alkyl substituents shifts their maximum absorption towards the red, as well as non-peripheral substitution. Bulky substituents are known to limit aggregation. Isobutyl moieties were selected to be introduced via thioether functions in one non-peripheral position of each of the four isoindole subunits of a Zn phthalocyanine. As shown in Scheme 1, the synthesis was readily achieved in two steps. First, phthalonitrile 1was prepared by the reaction between isobutylthiol and 3-nitrophthalonitrile, in 80% yield. The corresponding phthalocyanine Pc2 was obtained by reacting phthalonitrile 1in presence of Zn(OAc) 2 , yielding the desired phthalocyanine in 28% yield and in rather large scale (300 mg). All analyses confirmed the proposed structure. 3.2. Photo-properties of Pc2 UV-vis spectra of Pc2 have been recorded in THF, DMSO and chloroform at 10 micromolar, conditions for which Pc2 is not aggregated. DMSO has been selected as the solvent for the next measurements to use the unsubstituted ZnPc as the standard, as all necessary reference data are available. 33 The maximum of the Q band is located at 717 nm, reflecting the bathochromic shift effect expected from both the non-peripheral and the alkylthio substitution (Fig. 1A), compared to unsubstituted phthalocyanine that absorbs at 675 nm in DMSO 33 corresponding to a substantial bathochromic shift of more than 40 nm. The fluorescence spectrum recorded in DMSO showed the expected shape and small Stokes shift. The fluorescence quantum yield of Pc2 in DMSO was determined to be 0.07, in line with its high oxygen generation quantum yield (F D = 0.78). All the photo-properties of Pc2 in DMSO are summarized in Table 1. All these collected data confirm the relevance of using Pc2 for anti-cancer PDT, both for its strong absorption at far-red wavelength and its high ability to generate singlet oxygen. The fluorescence quantum yield of Pc2 is lower than those of ZnPc, while its SO quantum yield is higher. This is due to the presence of the sulfur atom on the phthalocyanine macrocycle, which is known to exert a heavy atom effect increasing the intersystem crossing. 35 3.3. Synthesis of RCP and BCP nanocarriers and encapsulation of Pc2. 3.3.1 Synthesis of RCP and BCP. Two kind of nanocarriers comprising hydrophilic oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA 300 ) and hydrophobic (2-acetoacetoxy)ethyl methacrylate (AEMA) repeat units were investigated: a random copolymer (RCP) and a block copolymer (BCP)(Scheme2).Due to their intrinsic amphiphilic nature, both can self-assemble in aqueous media in ultra-dilute conditions, yielding self-assembled structures whose topology can vary with the architecture of the starting copolymer. Both nanocarriers were synthesized via RAFT copolymerization of the commercially available monomers, using 4-cyanopentanoic acid dithiobenzoate (CPADB) as chain transfer agent (Scheme 2). After purification via precipitation in n-hexane and drying under dynamic vacuum, the isolated RCP and BCP were characterized by means of SEC/MALS and quantitative 1 H NMR Scheme 1 Synthesis of phthalocyanine Pc2 (only one of the existing regioisomers is shown). Paper Materials Advances Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 152 | Mater. Adv., 2025, 6, 148–156 © 2025 The Author(s). Published by the Royal Society of Chemistry spectroscopy for the determination of M n ,Ðand copolymer composition (see Table 2). The self-assembled blank nanoparticles (without phthalocyanine) SCNP-Pc2 0 (formed by RCP) and SLA-Pc2 0 (formed by BCP) at high dilution ([polymer] = 1 mg mL 1 ) in water were investigated by SAXS measurements. As illustrated in Fig. 2a, RCP in water at high dilution was found to form SCNP-Pc2 0 as determined by SAXS experiments. Due to the impossibility to perform SAXS experiments in the conditions that mimic the cell culture, SAXS experiments were performed in water in dilute conditions. Previous works by our group have confirmed the self-assembly of these amphiphilic random copolymers into globular like core–shell single-chain nanoparticles at a concentration well above the overlap concentration. 36,37 Concerning the use of the generalized Gaussian coil model to fit the SAXS data of RCP it is well-established in the literature. 25 The average radius of gyration (R g ) of the SCNPs was R g,water = 10.4 nm (a value smaller than that found in THF solvent, R g,THF = 14.4 nm). The SCNPs were found to have a sparse conformation as revealed by the high value of the size scaling exponent (n= 0.59). We attribute the open conformation of the SCNPs to the relatively small amount of hydrophobic AEMA units in RCP.On the other hand, BCP at high dilution in water self-assembled into star-like assemblies (SLA-Pc2 0 )ofR g,water = 10.0 nm having (on average) 2 arms each one (i.e., forming dimers). As we will see below, the number of arms per star increases upon encapsulation of Pc2. Concerning the SAXS data of BCP, the validity of the Dozier Star model to fit the SAXS data of star-like polymers is supported both by simulations and experimental Fig. 1 (A) Superimposed UV-vis spectra of Pc2 in chloroform, DMSO and THF (10 mM). (B) UV-vis absorption spectra of Pc2 in DMSO (2–12 mM). Inset: Absorption vs. concentration. (C) Absorbance, emission (irradiation: 640 nm), and excitation spectra of Pc2 in DMSO. (D) Determination of singlet oxygen quantum yield of Pc2 in DMSO (6 mM). Inset: Plot of DPBF absorbance at 417 nm vs. time. Table 1 Photo-properties of Pc2 in DMSO l max (nm) logel Em (nm) l Exc (nm) Dl Stokes (nm) F F F D 717 5.29 736 720 16 0.07 0.78 Scheme 2 (A) Pictorial representation of the copolymerization reaction of the hydrophobic monomer AEMA (depicted in grey) and the hydrophilic monomer OEGMA 300 (depicted in blue) to give RCP. (B) Reaction scheme for the synthesis of the amphiphilic block copolymer BCP. Table 2 Number average molecular weight (M n ), dispersity (Ð)and copolymer composition of RCP and BCP Copolymer M n /kDa ÐAEMA/mol% OEGMA 300 /mol% RCP 99.6 1.04 20 80 BCP 71.8 1.02 14 86 Materials Advances Paper Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 148–156 | 153 results. 38 It is worth noting that the UV-vis spectra of SCNP-Pc2 0 (formed by RCP) and SLA-Pc2 0 (formed by BCP) showed no absorption bands in the 600–700 nm wavelength region (Fig. S8, ESI†). 3.3.2. Encapsulation of Pc2. Both Pc2-containing SCNPPc2 x and SLA-Pc2 x were prepared using the same encapsulation procedure by starting with RCP or BCP, respectively. The experimental conditions for the encapsulation of Pc2 were first optimized exploring different polymer concentrations in solution. As explained above, we observed that the optimal working range for self-folding into SCNPs is from 0.1 mg mL 1 to 5 mg mL 1 for RCP and that BCP dimers start to form from a concentration of 1 mg mL 1 . Regarding the concentration of loadable Pc2, we looked for the highest achievable quantity of Pc2 to be compatible with the formation of SCNPs when using the less possible amount of RCP precursor, ensuring the highest possiblePc2/polymer ratio. This latter is a less flexible system, when compared to multi-chain aggregates, in terms of hydrophilic/hydrophobic interactions changes in the media, and constituted indeed our limiting factor for the optimization of the encapsulation procedure. We attempted the encapsulation fixing the photosensitizer concentration at 20 mM, which resulted in irreversible aggregation when using RCP as nanocarrier. For this reason, we progressively lowered the photosensitizer loading up to 12 mM, which finally resulted to be the maximum working Pc2 concentration. In addition, we selected the polymer concentration of 1 mg mL 1 as it is the lowest nanocarrier amount needed in solution to efficiently encapsulate the photosensitizer at its maximum achievable loading. To explore the effect of the loading amount, two different concentrations of Pc2 (x=5mMor12mM) were used for both copolymers, yielding for both the nanoparticles architectures (SCNPs or SLAs) the title phthalocyanine photosensitizer-encapsulated nanocarriers at the loading regimes of 4.7 mg per mg of polymer for SCNP-Pc2 5 and SLA-Pc2 5 and 11.2 mg per mg of polymer for SCNP-Pc2 12 and SLA-Pc2 12 . 3.3.3. Characterization of SCNP-Pc2 x .SAXS experiments confirmed the successful formation of SCNP-Pc2 5 and SCNPsPc2 12 , as illustrated in Fig. 3. Hence, a small reduction in size and in scaling exponent was observed upon increasing the amount of encapsulated Pc2. This may be because Pc2 is hydrophobic, so to encapsulate it the polymeric chain wraps the Pc2 complex, and this generates a compaction of the chain accompanied by a reduction in size. Table 3 shows the values of the fitting parameters for SAXS curves of SCNP-Pc2 0 (blank nanoparticles without phthalocyanine), SCNP-Pc2 5 and SCNPPc2 12 in water, applying a generalized Gaussian coil function. UV-vis spectra of SCNPs-Pc2 5 and SCNPs-Pc2 12 in water revealed an intense absorption in the 600–700 nm wavelength region (Fig. 4). The splitted shape of the Q band indicates that Pc2 is aggregated inside the nanocarrier. 3.3.4. Characterization of SLA-Pc2 x .SAXS results of SLAPc2 5 and SLA-Pc2 12 are illustrated in Fig. 5. Both nanocarriers in water at high dilution form star-like assemblies (SLAs) with a larger number of arms and slightly larger size than BCP (f=2 arms,R g,water = 10.0 nm). Analysis of the SAXS form factor of SLA-Pc2 5 in terms of the Dozier star model provided f= 4 arms with R g,water = 10.1 nm, whereas SLA-Pc2 12 displayed f= 8 arms and R g,water = 10.2 nm. Table 4 shows the values of the fitting parameters for SAXS curves of BCP,SLA-Pc2 5 and SLA-Pc2 12 in water, applying the Dozier star model. The characteristic UV-vis absorption bands of Pc2 encapsulated into SLA-Pc2 5 and SLA-Pc2 12 are reported in Fig. 6 and show the aggregated state of the phthalocyanine inside the nanoparticles. 3.4. In vitro photodynamic efficiency of SCNP-Pc2 x and SLA-Pc2 x The biological activity of the SCNP-Pc2 x and SLA-Pc2 x was studied on MCF-7 human breast cancer cells. Blank nanocarriers without Pc2 (RCP and BCP) were also tested to assess their own biocompatibility. Fig. 2 (A) SAXS results revealing the form factor of SCNP-Pc2 0 in water. Line is a fit of a generalized Gaussian coil function, yielding the values of the R g and nparameters. (B) SAXS results revealing the form factor of SLA-Pc2 0 in water. Line is a fit of the Dozier star model, yielding the values of the R g and fparameters. Fig. 3 SAXS results and respective depictions of the morphologies revealed, with form factor in water of SCNP-Pc2 5 (A) and SCNP-Pc2 12 (B),Values of R g and nwere obtained through fits of a generalized Gaussian coil function (continuous lines) to the experimental data. Paper Materials Advances Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 154 | Mater. Adv., 2025, 6, 148–156 © 2025 The Author(s). Published by the Royal Society of Chemistry 3.4.1. Cytotoxicity. First, the cytotoxic effect in the dark was analyzed by incubating the cells during 72 h with increasing concentrations of SCNP-Pc2 x or SLA-Pc2 x (from 1 to 200 mgmL 1 ). The corresponding concentrations of Pc2 for each nanocarrier were calculated (in mgmL 1 and in mM) and reported in the Table S1 (ESI†). The cells were submitted to a MTT colorimetric assay, and the quantification of living cells was performed (Fig. 7A). All the nanocarriers, either empty or phthalocyanine-loaded, induced no more than 50% cell death, even at the highest concentrations (200 mgmL 1 ). This observation let us to fix a concentration of 100 mgmL 1 for PDT experiments. 3.4.2. Photodynamic therapy. Next, to evaluate the photodynamic potential of the Pc2-loaded nanocarriers, MCF-7 cells were incubated with 100 mgmL 1 of distinct nanocarriers loaded with 5 or 12 mM of phthalocyanine (i.e.,SCNP-Pc2 5 , SCNP-Pc2 12 ,SLA-Pc2 5 and SLA-Pc2 12 ) for 24 h and exposed or not to laser irradiation at various wavelengths (633, 650 and 740 nm). These wavelengths correspond to the absorption range of Pc2 when encapsulated into the nanocarriers, as its Q band is enlarged due to their aggregated state inside the nanocarrier. Living cell percentage quantification was performed 2 days after irradiation (Fig. 7B). SCNP-Pc2 5 and SCNP-Pc2 12 did not exhibit significant phototoxicity, while SLA-Pc2 5 and SLA-Pc2 12 resulted in significant efficiency. SLAPc2 12 excited at 650 nm and SLA-Pc2 5 excited at 740 nm gave the best results with 47% and 73% cell death, respectively. When comparing the absorption spectra of SCNP-Pc2 5 with SCNP-Pc2 12 (as well as for SLA-Pc2 5 vs.SLA-Pc2 12 ), no significant difference was observed. For this reason, we ascribed the augmented PDT-effect in SLA-Pc2 x to the more flexible structure and peculiar topology of these BCP-based nanocarriers that accommodate Pc2 in a manner favoring likely better ROS generation. In parallel, we have verified the cytotoxicity and PDT potential of free Pc2. For this, MCF-7 cells were incubated Table 3 Fitting parameters values for SAXS curves of SCNP-Pc2 0 ,SCNPPc2 5 and SCNP-Pc2 12 in water, applying a generalized Gaussian coil function SCNP-Pc2 0 SCNP-Pc2 5 SCNP-Pc2 12 R g (nm) 10.4 10.1 9.4 n0.60 0.56 0.53 I 0 0.048 0.053 0.053 Fig. 4 UV-vis spectra of SCNP-Pc2 5 and SCNP-Pc2 12 in water (1 mg mL 1 ). Fig. 5 SAXS results and respective depictions of the morphologies revealed, with form factor in water of SLA-Pc2 5 (A) and SLA-Pc2 12 (B). Values of R g and arm number (f) were obtained through fits of the Dozier star model to the experimental data (continuous lines). Table 4 Fitting parameters values for SAXS curves of SLA-Pc2 0 ,SLA-Pc2 5 and SLA-Pc2 12 in water, applying Dozier star model SLA-Pc2 0 SLA-Pc2 5 SLA-Pc2 12 R g (nm) 10.0 10.1 10.2 f248 n0.60 0.60 0.60 I 0 0.040 0.052 0.068 Fig. 6 UV-vis spectra of SLA-Pc2 5 and SLA-Pc2 12 in water (1 mg mL 1 ). Materials Advances Paper Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 148–156 | 155 with increasing concentrations of Pc2 and didn’t show any toxicity for these concentrations (Fig. S11A, ESI†). Cells were then incubated with 0.5 or 1.2 mgmL 1 of Pc2 (corresponding to 100 mgmL 1 of SCNP-Pc2 5 ,SCNP-Pc2 12 ,SLA-Pc2 5 ,SLA-Pc2 12 ) and submitted to 633 nm, 650 nm and 740 nm excitation. Data demonstrated an absence of PDT potential without encapsulation (Fig. S11B, ESI†). This result highlights the necessity of Pc2 encapsulation for a biological application. In addition, reactive oxygen species (ROS) production was detected under light excitation at 650 nm when cells were incubated with SLAPc2 12 but not with free Pc2 (1.2 mgmL 1 ) (Fig. S12, ESI†). It is well-known that aggregated photosensitizers - including phthalocyanines - have a self-quenched inter-system crossing pathway, negatively affecting their photodynamic efficiency when excited classically by monophotonic light source. On the other hand, aggregated phthalocyanines, as in the case of phthalocyanine-based organosilica nanoparticles previously reported by some of us, 20,21 may exhibit good photo-killing effect when excited by pulsed laser at NIR wavelengths. The photodynamic efficiency of SCNP-Pc2 5 ,SCNP-Pc2 12 ,SLA-Pc2 5 and SLAPc2 12 , still at 100 mgmL 1 , was therefore studied with a pulsed laser irradiating at 800 nm (Fig. 7C) in the first NIR phototherapeutic window. Under such conditions, SCNP-Pc2 5 resulted to be the only nanoparticle to exhibit significant photo-killing ability. Indeed, we can observe 36% cell death when cells, previously incubated with SCNP-Pc2 5 , were submitted to 800 nm excitation wavelength using a pulsed laser, from Chameleon lamp of a confocal microscope (3 successive scans of 1.5 s). The comparison between SCNP-Pc2 5 (4.7 mg per mg of polymer) and its higher-Pc2loaded analogue SCNP-Pc2 12 (11.2 mg per mg of polymer) shows that here also the loading is important, as too much phthalocyanine in the SCNP carrier prevents a good efficency. Overall, the different behavior of these phthalocyanine-loaded nanocarriers can be attributed to their different nanocarrier topology: selffolded single-chain nanoparticles (SCNPs)vs. star-like aggregates (SLAs), respectively. 4. Conclusions Two different amphiphilic copolymers encapsulating Pc2 within self-folded single-chain nanoparticles (SCNPs) and star-like aggregates (SLAs), have demonstrated their relevance as photosensitizer nanocarriers for PDT of cancer cells, under excitation in the infrared range with pulsed laser or in the visible range with continuous laser, respectively. In addition, free Pc2 is not biologically active in these conditions. All together, these results demonstrate the interest and the novelty of these nanocarrier systems for the encapsulation and delivery of photosensitizers for PDT. Author contributions DA, MO, CN, ZS-: data curation, investigation, formal analysis and manuscript writing. U ¨I ˙, EVS, AI, AA: data curation, supervision, formal analysis, methodology and manuscript writing. FD, JAP and MGB: supervision, conceptualization, resources, project administration and manuscript writing. Data availability The data supporting this article have been included as part of the ESI.† Conflicts of interest There are no conflicts to declare. Fig. 7 (A) Cell viability study of MCF-7 cells incubated 72 h with increasing concentrations (from 1 to 200 mgmL 1 )ofSCNP-Pc2 5 ,SCNP-Pc2 12 ,SLAPc2 5 and SLA-Pc2 12 . (B) PDT effect of SCNP-Pc2 5 ,SCNP-Pc2 12 ,SLA-Pc2 5 and SLA-Pc2 12 (100 mgmL 1 ) incubated 24 h with MCF-7 cells and excited at 633, 650 and 740 nm. (C) PDT effect of SCNP-Pc2 5 ,SCNP-Pc2 12 ,SLAPc2 5 and SLA-Pc2 12 (100 mgmL 1 ) incubated 24 h with MCF-7 cells and excited at 800 nm, with a pulsed laser. Data are means SEM; n=3.*po 0.05, significantly different from ‘‘No laser’’. Paper Materials Advances Open Access Article. Published on 05 November 2024. Downloaded on 2/7/2025 3:22:40 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online