Synthesis, Photo-Characterizations, and Pre-Clinical Studies on Advanced Cellular and Animal Models of Zinc(II) and Platinum(II) Sulfonyl-Substituted Phthalocyanines for Enhanced Vascular-Targeted Photodynamic Therapy

Two phthalocyanine derivatives tetra-peripherally substituted with tert-butylsulfonyl groups and coordinating either zinc(II) or platinum(II) ions have been synthesized and subsequently investigated in terms of their optical and photochemical properties, as well as biological activity in cellular, tissue-engineered, and animal models. Our research has revealed that both synthesized phthalocyanines are effective generators of reactive oxygen species (ROS). PtSO2tBu demonstrated an outstanding ability to generate singlet oxygen (ΦΔ = 0.87–0.99), while ZnSO2tBu in addition to 1O2 (ΦΔ = 0.45–0.48) generated efficiently other ROS, in particular ·OH. Considering future biomedical applications, the affinity of the tested phthalocyanines for biological membranes (partition coefficient; log Pow) and their primary interaction with serum albumin were also determined. To facilitate their biological administration, a water-dispersible formulation of these phthalocyanines was developed using Pluronic triblock copolymers to prevent self-aggregation and improve their delivery to cancer cells and tissues. The results showed a significant increase in cellular uptake and phototoxicity when phthalocyanines were incorporated into the customizable polymeric micelles. Moreover, the improved distribution in the body and photodynamic efficacy of the encapsulated phthalocyanines were investigated in hiPSC-delivered organoids and BALB/c mice bearing CT26 tumors. Both photosensitizers exhibit strong antitumor activity. Notably, vascular-targeted photodynamic therapy (V-PDT) led to complete tumor eradication in 84% of ZnSO2tBu and 100% of PtSO2tBu-treated mice, and no recurrence has so far been observed for up to five months after treatment. In the case of PtSO2tBu, the effect was significantly stronger, offering a wider range of light doses suitable for achieving effective PDT.


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with an optical path of 1 cm.The molar absorption coefficients were determined from Beer's law.Fluorescence spectra were recorded using a Perkin Elmer LS 55 Fluorescence Spectrometer, with a slit width of 8 nm and a scan rate of 100 nm/min.The fluorescence spectra were recorded at an excitation radiation wavelength of 592 nm.The fluorescence lifetimes of the compounds were recorded with a Fluorolog-3 Spectrometer (Horiba Jobin-Yvon) using Time-Correlated Single Photon Counting (TCSPC).Measurements were carried out using a picosecond pulsed diode with a wavelength of 372 nm and for an excitation pulse duration of 200 ns.Fluorescence decay curves for each compound were recorded at the wavelength corresponding to its emission maximum, and the maximum number of counts per peak channel was 10000.Instrument response function (IRF) was obtained by using a standard, aqueous colloidal silica solution, LUDOX.The collected data were analyzed using Horiba Jobin-Yvon's DAS6 v6.4 program.Two-exponential models were fitted to measure fluorescence decay over time for phthalocyanines in THF, so that CHiQ values were in the range of 1.2-1.4,and residuals were symmetric about the zero axis.
Photodegradation tests.The photodegradation process of phthalocyanines was evaluated by spectrophotometric method using an Infinite M200 microplate reader from Tecan.Solutions of photosensitizers in THF:PBS (1:99) with TRITON X-100 were exposed using a 635±20 nm laser diode as a light source.The irradiance to which the test solutions were exposed was 17 mW/cm 2 and was monitored using a handheld NOVA II laser power and energy meter from OPHIR.During the irradiation of the samples, a cut-off < 550 nm filter was used to eliminate radiation below 550 nm.During the irradiation, the absorbance value at the Q-band maximum of the photosensitizer solutions was measured after successively increasing time intervals.
Determination of singlet oxygen quantum yields (ΦΔ).The quantum yield of singlet oxygen generation by phthalocyanines in DMF was determined by an indirect method involving chemical quenching using 1,3-diphenylisobenzofuran (DPBF) as the acceptor 1 O2.The values of ΦΔ were determined by the comparative method, using the relation (1): Where ΦΔ and ΦΔ(Std) are the quantum yields of singlet oxygen generation for the sample and standard, RStd and R are the photobleaching rates of the standard and phthalocyanine, respectively, and IStd and I are the absorbance intensities of the standard and test compound.A commercially available zinc phthalocyanine with known singlet oxygen generation quantum yield (ΦΔ=0.56 in DMF) was used as a standard.Working solutions were prepared, one of which was a 6 μM solution of DPBF in DMF (A ~1.3), while the other was a 5 μM solution of the corresponding phthalocyanines.The procedure consisted of irradiating the compound mixture placed in a quartz cuvette at a volume ratio of 1:1 with a xenon lamp (XBO 150) in the presence of a cut-off filter < 550.The irradiance, monitored with a handheld NOVA II laser power and energy meter from OPHIR, was ~67 mW/cm 2 .The singlet oxygen generating capacity of phthalocyanines was evaluated by changing the absorbance intensity of the acceptor band maximum 1 O2 (414 nm) during sample irradiation.The value of singlet oxygen generation efficiency for ZnSO2tBu and PtSO2tBu was also determined based on stationary measurements using the single-point method.For this purpose, solutions of the standard (phenalenone) and the test substances in DMF were prepared so that their absorbances at the wavelength of the excitation radiation were equal.For the prepared solutions, singlet oxygen emission spectra were recorded at the excitation light wavelength λex= 350 nm (absorbance ~0.20), using a cut-off filter < 715 nm.Measurements were made with a Fluorolog-3 spectrophotometer (Horiba Jobin-Yvon) equipped with a LATERAL 5509 detector cooled with liquid nitrogen for near-infrared (NIR) measurements.Considering the correction for the slight difference in absorbance at λex, the values of ΦΔ were determined based on the relation (2), where: ΦΔ and ΦΔ(Std) the quantum yield of singlet oxygen generation for the test sample and standard, Area ( ) oraz Area ( Lewis Lung Carcinoma (LLC) and human breast cancer cells (MCF-7) were cultured in RPMI1640 medium.All media were supplemented with 10% of FBS and 1% of antibiotics.
Prior to the experiments, the cells were detached using trypsinization, seeded in microplates, and maintained in a humidified environment at 37°C with 5% CO2.
PS accumulation in organoids.The organoids were subjected to a 24-hour incubation with photosensitizers solutions at a concentration of 1.5 mg/kg BW.Following this incubation period, the organoids were washed two times with PBS, and Hoechst33342 was introduced to the samples for 15 minutes.Subsequently, the samples were rinsed twice with HBSS and made ready for visualization.The imaging process was conducted using a Zeiss LSM 880 confocal microscope (Carl Zeiss, Jena, Germany) equipped with a 40× immersion objective.The resulting images were captured and analyzed utilizing Zeiss ZEN software.
Animal model.The mean size of the tumors were calculated according to the following formula (3):
Analysis.The STATISTICA software for biostatistics (StatSoft Inc.) was used for statistical analysis of the data.The data are expressed as means ± standard deviation or standard error of the mean (SEM) of at least three independent experiments.Statistical significance was determined by one-way or two-way ANOVA with Bonferroni post hoc test using GraphPad Prism version 5.0.0 for Windows, GraphPad Software, San Diego, California USA.

Figure S1
Figure S1 Electronic absorption and fluorescence spectra of ZnSO2tBu and PtSO2tBu

FigureFigure S3 Figure S4 Figure S5
Figure S2 a) Distribution of the normalized weighted difference function for individual

Figure
Figure S6 a) Emission spectra of phthalocyanines in different phases (n-octanol, PBS), b)

Figure S10
Figure S10The red pixels quantification based on the images registered for organoids after

Figure S12
Figure S12Observations of changes in tumor 1 day after, 7 days after and 18 days after a) ZnPc