Topology effects in photodynamic therapy with phthalocyanine nanocarriers (2024)

Davide Arena^a, Ümit İşci^b, Mélanie Onofre^c, Christophe Nguyen^c, Zeynel Şahin^b, Ester Verde-Sesto^ad, Amaia Iturrospe^a, Arantxa Arbe^a, Magali Gary-Bobo*^c, José A. Pomposo*^ade and Fabienne Dumoulin*^f
aCentro de Física de Materiales (CSIC, UPV/EHU) and Materials Physics Center MPC, Paseo Manuel Lardizabal 5, 20018 Donostia, Spain. E-mail: josetxo.pomposo@ehu.eus
^bMarmara University, Faculty of Technology, Department of Metallurgical and Materials Engineering, 34854 Maltepe, Istanbul, Türkiye
^cInstitut des Biomolécules Max Mousseron, CNRS, ENSCM, 34293 Montpellier, France. E-mail: magali.gary-bobo@inserm.fr
^dIKERBASQUE–Basque Foundation for Science, Plaza de Euskadi 5, 48009 Bilbao, Spain
^eDepartamento de Polímeros y Materiales Avanzados, Física, Química y Tecnología, University of the Basque Country (UPV/EHU), 20800 Donostia, Spain
^fAcıbadem Mehmet Ali Aydınlar University, Faculty of Engineering and Natural Sciences, Department of Biomedical Engineering, 34752 Ataşehir, Istanbul, Türkiye. E-mail: fabienne.dumoulin@acibadem.edu.tr

Received 21st May 2024, Accepted 5th November 2024

First published on 5th November 2024

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 anti-cancer 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 nano-formulated photosensitizers can benefit from the enhanced permeability and retention (EPR) effect, which may have some limitations14 but is anyway a good way to deliver photosensitizers. 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 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 CO₂-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 self-assembled 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 small-angle 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 (OEGMA₃₀₀) (99%), (2-acetoacetoxy)ethyl methacrylate (AEMA) (95%), 2,2′-azobis(2-methylpropionitrile) (AIBN) (≥98%), 1,4-dioxane, n-hexane, deuterated chloroform (CDCl₃) (99.96 atom% D, containing 0.03% (v/v) tetramethylsilane, TMS), sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O), sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O) were purchased from Sigma-Aldrich and used, unless specified, as received. 4-Cyanopentanoic acid dithiobenzoate (CPADB) (≥97%) was purchased from Strem Chemicals. Methanol and tetrahydrofuran (THF) (HPLC grade) were purchased from Scharlab. AIBN was recrystallized from methanol. OEGMA₃₀₀ and AEMA were purified by passing through basic alumina. 3-Nitrophthalonitrile, isobutylthiol, hexadeuterated dimethylsulfoxide (DMSO-d₆), dimethylformamide (DMF), potassium carbonate (K₂CO₃), 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 CDCl₃ or DMSO-d₆ solutions on a Varian spectrometer (500 MHz for ¹H, 125 MHz for ¹³C 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 °C 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). ¹H NMR (500 MHz, CDCl₃) δ, ppm: 7.61–7.56 (m, 3 H), 2.96 (d, 2H), 1.96 (m, 1 H), 1.11 (d, 6 H). ¹³C NMR (125 MHz, CDCl₃) δ, 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 (ν, 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 C₄₈H₄₈N₈S₄Zn: 930.588. FT-IR (ν, cm⁻¹) 3059, 2953, 1592, 1565, 1462, 1430, 1365, 1330, 1311, 1222, 1163, 1103, 1041, 946, 900, 881, 792, 757, 735, 679. ¹H NMR (500 MHz, DMSO-d₆) δ, 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): λ_max, nm (logε): 710 (5.3), 641 (4.6), 340 (4.9).

2.1.4. Synthesis of the random copolymer poly(OEGMA₃₀₀-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 OEGMA₃₀₀, 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 °C, 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. ¹H NMR (400 MHz, CDCl₃) δ, ppm: 4.37 (m, CCO₂CH₂CO₂CH₂), 4.18–4.11 (m, 2H, CCO₂CH₂CO₂), 3.83–3.58 (m, CH₂OCH₂), 3.41 (s, O), 2.33 (s, CH₂CO), 1.81 (m, CCH₂), 1.04 (m, CCH₃), 0.87 (m, CCH₃).

2.1.5. Synthesis of the block copolymer poly(OEGMA₃₀₀)-b-poly(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 °C for 5 hours. Then, the reaction was quenched by freezing the mixture at −78 °C. 1 mL (0.014 mmol of macro-CTA) of the crude, 6 mL (21 mmol) of OEGMA₃₀₀, 2.3 mg (0.014 mmol) of AIBN and 18 mL of 1,4-dioxane were degassed purging nitrogen for 20 minutes, then left stirring at 70 °C under positive nitrogen pressure for 17 hours. After this time, the reaction was quenched by cooling the mixture at −78 °C. 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. ¹H NMR (400 MHz, CDCl₃) δ, ppm: 4.37 (m, CCO₂CH₂CO₂CH₂), 4.18–4.10 (m, 2H, CCO₂CH₂CO₂), 3.77–3.57 (m, CH₂OCH₂), 3.40 (s, O), 2.32 (s, CH₂CO), 1.87 (m, CCH₂), 1.04 (m, CCH₃), 0.87 (m, CCH₃).

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 μmol) 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 μL or 447 μL). 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 μM, 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. SCNP-Pc2_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:

where F and F_Std are the areas under the emission curves for the sample and standard, respectively. A and A_Std are the absorbance at the excitation wavelength for the sample and standard, n is the refractive index of the solvents for the sample and reference. Unsubstituted ZnPc in DMSO was used as a standard (ΦF_(Std) = 0.20) and sample and standard were excitated (at λ= 640 nm) at the same wavelength in DMSO.

Singlet oxygen quantum yield of Pc2 was obtained in DMSO solution (in air, no oxygen bubbled) by indirect method with zinc phthalocyanines (ZnPc) as a reference and 1,3-diphenylisobenzofuran (DPBF) as a chemical quencher for singlet oxygen, using the fallowing equation:

where is the singlet oxygen quantum yield for the standard ZnPc (Φ_Δ = 0.67 in DMSO). R and 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 (∼6 μM, absorption ∼1.2 at 717 nm) and DPBF (∼30 μM, absorption ∼0.8 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 Φ_Δ 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 °C, in humidified atmosphere and under 5% CO₂.

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 μg mL⁻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 μL of an equal volume 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 μg mL⁻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) was performed with an EVOS 5000 microscope using Cy5 EVOS LED light cube during 1 min, 64,9 mW at magnification 4×. The excitation at 650 nm (39 J.cm⁻²) was performed with a portable laser during 20 min. The excitation at 740 nm (11.25 J m⁻²) was performed with a LED spotlight from EvoluChem™ for 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 μg mL⁻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 (Carl Zeiss 10-fold magnification/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 p value < 0.05 was considered as statistically significant.

3. Results and discussion

3.1. Molecular design and synthesis of iso-butylthio non-peripherally 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 1 was prepared by the reaction between isobutylthiol and 3-nitrophthalonitrile, in 80% yield. The corresponding phthalocyanine Pc2 was obtained by reacting phthalonitrile 1 in 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 DMSO33 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 (Φ_Δ = 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

Table 1 Photo-properties of Pc2 in DMSO

λmax (nm)	logε	λEm (nm)	λExc (nm)	ΔλStokes (nm)	ΦF	ΦΔ
717	5.29	736	720	16	0.07	0.78

3.3. Synthesis of RCP and BCP nanocarriers and encapsulation of Pc2.

3.3.2. Encapsulation of Pc2. Both Pc2-containing SCNP-Pc2_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 μM, which resulted in irreversible aggregation when using RCP as nanocarrier. For this reason, we progressively lowered the photosensitizer loading up to 12 μM, 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 = 5 μM or 12 μM) 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 μg per mg of polymer for SCNP-Pc2₅ and SLA-Pc2₅ and 11.2 μg per mg of polymer for SCNP-Pc2₁₂ and SLA-Pc2₁₂.

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.

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, ZŞ: data curation, investigation, formal analysis and manuscript writing. Üİ, 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.

Acknowledgements

D. A., E. V.-S., A. I., A. A. and J. A. P. gratefully acknowledge Grant PID2021-123438NB-I00 funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”; Grant TED2021-130107A-I00 funded by MCIN/AEI/10.13039/501100011033 and Unión Europea “NextGenerationEU/PRTR”, Grant IT-1566-22 from Eusko Jaurlaritza (Basque Government), and financial support from RyC program (RYC2022-037590-I). We acknowledge the imaging facility MRI, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04, “Investments for the future”).

Notes and references

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