Accomplishment of α -Chymotrypsin on Photodynamic Effect of Octa-Substituted Zn(II)- and Ga(III)-Phthalocyanines against Melanoma Cells

: Octa-methylpyridiloxy-substituted Zn(II)- and Ga(III)-phthalocyanines ( ZnPc1 and GaPc1 ) were studied on human pigmented melanoma (SH4) and keratinocyte (HaCaT) cell lines. The efficacy of ZnPc1 and GaPc1 against melanoma cells was compared to the results in the presence of a protease α -chymotrypsin (ChT). The synthesis and characterization of compounds were carried out using well-known approaches. The formation of physical conjugates due to the addition of ChT was studied via absorption and fluorescence. The proteolytic activity of ChT was verified with casein as a substrate. The photosafety of compounds was proven on embryonal cells (BALB 3T3) under solar exposure (LED 360–1100 nm). The photodynamic activity of GaPc1 and ZnPc1 was studied for a concentration range of irradiation (LED 660 nm). The reduction of the proteolytic activity of ChT was observed only for the irradiation of ZnPc1 or GaPc1 . GaPc1 and ChT and their conjugates, except ZnPc1 (PIF ~6), were evaluated as photo-safe to solar light (PIF < 2). The efficiency of GaPc1 was shown to be much higher than that of ZnPc1 in their individual applications. The phototherapeutic index of GaPc1 (PI = 1.71) on SH4 cells was higher for the conjugate. α - Chymotrypsin and phthalocyanine have the advantages of reducing high toxicity and increasing the phototherapeutic index.


Introduction
Melanoma continues to be a serious threat to human health.Despite the scientific advancements in chemical science and the current pharmaceutical novelties, existing melanoma treatments still have many unfavorable side effects and low efficiency [1].Photodynamic therapy (PDT) has emerged as a tolerable clinical procedure for localized tumors [2].The method is based on the production of cytotoxic oxygen species in the spot of irradiation on the tumor area, avoiding the cytotoxic effect on surrounding healthy cells and the whole body.PDT involves the administration of a photosensitizer (PS), its accumulation in the malignant cells, and irradiation with light of specific wavelengths and properties to activate the PS in a triplet excited state molecule that produces a variety of reactive oxygen species (ROS) that induce cell death [3].The potential of PDT against tumors with no other alternative treatment options is well admitted [4].Bioactive antibody molecules, drug-delivery biomolecules, and other tumor-specific biomolecules are well known for targeting PDT [5].
Phthalocyanine complexes (MPcs) are heterocyclic macromolecules that are identifiable as second-generation PSs for PDT because of their unique photo-properties that can be modified by their easy-to-tailor structure, which facilitates the achievement of compatible properties for PDT [6].Despite their promising photochemistry, the MPc-photosensitizers feature several complications for clinical applications, such as undesirable dark toxicity, slow body clearance, and a lack of water solubility, which necessitate further studies for improvement [7].
Proteolytic enzymes take part in the chemical reaction of hydrolysis of peptide bonds and in bio-synthesis and are used for cell detachment in practice.Serine proteases, among which is α-chymotrypsin (ChT), have been evaluated for their promising potential as anticancer agents [8].A recent study reported that nano-particles with a protease can be powerful medical tools to slow chemoresistance [8].It was found that therapy that includes proteases may improve drug bioavailability, reduce circulation time, and increase the selective uptake in tumors [9].These necessities arise due to the need for lower drug doses for body protection, a lack of side effects, and a better quality of life [10].The human body produces proteins and enzymes that may serve as natural drug vehicles in circulation, as well as for specific carriers of anticancer drugs [11].A promising approach based on the different mechanisms of action of different molecular species for targeted PDT was reported [12].Another option is the possibility of obtaining quenched PS-conjugates to restore fluorescence emission and activate the generation of ROS after protease digestion [13].The role of protease activity was exploited in drug delivery for the site-specific release of chemo-drugs at the tumor and their rapid activation by the target protease [14].The results of preclinical studies with protease-sensitive prodrugs or fluorescence imaging agents suggested the prospect for the next clinical estimation of site-selective prodrugs or activators [15].In addition to the wide-ranging usage of enzymes in proteins and cell research, they are well-accepted as catalysts in clinical diagnostics and routine therapeutic practice [16].
Enzyme-activated PDT drugs were evaluated without dependence on the enzyme function because of the main mechanism of singlet oxygen generation that directly destroys malignant cells [17].Generally, proteases are part of a wide variety of diseases, and their importance to sickness pathology makes them excellent therapeutic targets.An initial idea of protease-sensitive prodrugs, also referred to as PDT action, has the key goal of increasing PS uptake [18].The conjugation was shown to lower the fluorescence quantum yield, triplet state quantum yield, and ability for photosensitivity [19].This study reported that enzymes reduced the singlet oxygen quantum yield of pheophorbides (from 0.52 to 0.17 and 0.04).Another study reported the development of a strategy termed proteasemediated PDT (PM-PDT) with an activation step from proteases on PS and a positive effect on PDT due to proteases' catalytic activity [20].Some examples are serine endoproteinases, trypsin, and chymotrypsin, which were studied and found to have promising therapeutic efficacy against cancer [21].Among the proteolytic enzymes, only ChT in combination with trypsin has been approved as a medication for clinical usage since the early 1960s [22].Proteases are plentiful and sufficient for several diseases, so these inhibitory therapies are often used in cure regimes together with other cancer therapies.Chen and co-authors [23] offered a first illustration of synergistically destroying the extracellular matrix (ECM) by combining digestive enzymes with the generation of reactive oxygen species.This approach of combining ChT as an enzyme with oxygen species generated during PDT showed a tendency to improve the uptake and penetration of drugs into tumors.The acidity of tumors, plus the sufficient singlet oxygen due to PDT, can enhance their efficacy because of the enzymes that catalyze protein decomposition.Thus, it has resulted in effective damage to the tumor matrix and increased the therapeutic effect.The incorporation of proteases permitted a better permeation of the drug in cancer tissues and the ability for more efficient singlet oxygen saturation than for the free-state molecule [24].
The present study aims to investigate the influence of the protease α-Chymotrypsin on the PDT efficacy of two phthalocyanine complexes (ZnPc1 and GaPc1) against human pigmented melanoma (SH-4) in comparison to normal keratinocyte cells (HaCaT).The effect of the macromolecule of ChT on phthalocyanines' photophysical properties was studied in a buffer solution (pH 7.8).The photosafety of compounds was analyzed on a fibroblast cell line (BALB/3T3) under artificial solar light exposure.The proteolytic activity of ChT was tested in a prior in vitro study, and the inhibition ability of the complexes (ZnPc1 and GaPc1) was proven after irradiation with LED 660 nm and UV 254 nm spectra.The PDT efficiency of both phthalocyanines applied alone and with the addition of the protease chymotrypsin was studied in a concentration-dependent and comparison-based manner for a set of light parameters.

Synthesis
Zn(II)-phthalocyanine (ZnPc1) with eight peripheral methylpyridiloxy substitution groups was successfully synthesized following a slightly modified well-known procedure summarized in Scheme 1.The previous synthetic procedure of cyclotetramerization was applied [25,26].The synthetic procedure starts from commercial dinitrile 1.It includes a nucleophilic displacement reaction to obtain dinitrile 2. Direct cyclotetramerization and a multiple-step procedure were used to obtain ZnPc1.A metal-free phthalocyanine is a favorable starting compound for phthalocyanine complexes and facilitates the production of high-purity complexes.The metalation was conducted with zinc acetate as a metal salt.The complex of gallium (GaPc1) was possible to obtain only at a high temperature (~220 • C) in the presence of a catalyst (DBU) starting from a monomer [27].Both metal complexes (ZnPc1 and GaPc1) were isolated from the reaction mixture by precipitation in hexane and washed with several solvents.The purification was carried out by column chromatography on silica gel with an eluent mixture of dichloromethane (DCM) and methanol (DCM:MeOH, 9:1).All compounds were characterized by analytical methods.
tissues and the ability for more efficient singlet oxygen saturation than for the free-state molecule [24].
The present study aims to investigate the influence of the protease α-Chymotrypsin on the PDT efficacy of two phthalocyanine complexes (ZnPc1 and GaPc1) against human pigmented melanoma (SH-4) in comparison to normal keratinocyte cells (HaCaT).The effect of the macromolecule of ChT on phthalocyanines' photophysical properties was studied in a buffer solution (pH 7.8).The photosafety of compounds was analyzed on a fibroblast cell line (BALB/3T3) under artificial solar light exposure.The proteolytic activity of ChT was tested in a prior in vitro study, and the inhibition ability of the complexes (ZnPc1 and GaPc1) was proven after irradiation with LED 660 nm and UV 254 nm spectra.The PDT efficiency of both phthalocyanines applied alone and with the addition of the protease chymotrypsin was studied in a concentration-dependent and comparison-based manner for a set of light parameters.

Synthesis
Zn(II)-phthalocyanine (ZnPc1) with eight peripheral methylpyridiloxy substitution groups was successfully synthesized following a slightly modified well-known procedure summarized in Scheme 1.The previous synthetic procedure of cyclotetramerization was applied [25,26].The synthetic procedure starts from commercial dinitrile 1.It includes a nucleophilic displacement reaction to obtain dinitrile 2. Direct cyclotetramerization and a multiple-step procedure were used to obtain ZnPc1.A metal-free phthalocyanine is a favorable starting compound for phthalocyanine complexes and facilitates the production of high-purity complexes.The metalation was conducted with zinc acetate as a metal salt.The complex of gallium (GaPc1) was possible to obtain only at a high temperature (~220 °C) in the presence of a catalyst (DBU) starting from a monomer [27].Both metal complexes (ZnPc1 and GaPc1) were isolated from the reaction mixture by precipitation in hexane and washed with several solvents.The purification was carried out by column chromatography on silica gel with an eluent mixture of dichloromethane (DCM) and methanol (DCM:MeOH, 9:1).All compounds were characterized by analytical methods.Scheme 1. Synthesis of octa-substituted Zn(II)-phthalocyanine (ZnPc1).The same pathway was applied for Ga(III)-phthalocyanine (GaPc1).Scheme 1. Synthesis of octa-substituted Zn(II)-phthalocyanine (ZnPc1).The same pathway was applied for Ga(III)-phthalocyanine (GaPc1).

Spectrophotometric Study
Spectrophotometric studies of cationic complexes (ZnPc1 and GaPc1) were performed in a comparison-based manner in phosphate buffer (PB, pH 7.8) and in DMSO solutions at room temperature Figure 1a).The absorption spectra in the visible region presented the typical Q-bands of the monomeric compound for GaPc1 and the molecular associates for ZnPc1 in water solutions.The maxima were recorded at 681 nm (DMSO) and 678 nm (PB) for GaPc1, assigned by π-π* transitions.The complex ZnPc1 showed a Q-band at 676 nm (DMSO) and the split and a low-intensity Q-band (640 nm and 672 nm) in an aqueous solution due to aggregation.This observation is associated with the formation of J-aggregates of phthalocyanine molecules.A wide B-band was recorded between 328 nm and 395 nm for ZnPc1 (DMSO), and there was a low-intensity vibrionic band in the visible region with a peak at 615 nm (ZnPc1) and 612 nm (GaPc1).The recorded Q-band maxima of both compounds showed that eight substituents to the Pc-ring molecule slightly shifted the Q-band to the red spectrum region.Both are relatively similar to the peaks of similar peripheral tetra-substituted Ga(III) or Zn(II) phthalocyanine complexes [28][29][30].

Spectrophotometric Study
Spectrophotometric studies of cationic complexes (ZnPc1 and GaPc1) were performed in a comparison-based manner in phosphate buffer (PB, pH 7.8) and in DMSO solutions at room temperature Figure 1a).The absorption spectra in the visible region presented the typical Q-bands of the monomeric compound for GaPc1 and the molecular associates for ZnPc1 in water solutions.The maxima were recorded at 681 nm (DMSO) and 678 nm (PB) for GaPc1, assigned by π-π* transitions.The complex ZnPc1 showed a Q-band at 676 nm (DMSO) and the split and a low-intensity Q-band (640 nm and 672 nm) in an aqueous solution due to aggregation.This observation is associated with the formation of J-aggregates of phthalocyanine molecules.A wide B-band was recorded between 328 nm and 395 nm for ZnPc1 (DMSO), and there was a low-intensity vibrionic band in the visible region with a peak at 615 nm (ZnPc1) and 612 nm (GaPc1).The recorded Q-band maxima of both compounds showed that eight substituents to the Pcring molecule slightly shifted the Q-band to the red spectrum region.Both are relatively similar to the peaks of similar peripheral tetra-substituted Ga(III) or Zn(II) phthalocyanine complexes [28][29][30].The absorption spectra were recorded during the titration of the solutions of the complexes (ZnPc1 and GaPc1) in PB, pH 7.8 with ChT for a concentration range of 5.12-50.6µM ChT (Figure 1b,c).The Q-band of the aggregated ZnPc1 was not changed by an increase of ChT, suggesting that the compound was not changed.The spectra show two isosbestic points at 325 nm and 375 nm (GaPc1) and at 304 nm and 406 nm (ZnPc1), which are most likely due to the dilution for the spectra in the visible region and the increase in the UV-vis region due to additional aromatic amino acids (due to ChT).The addition of GaPc1 in a concentration range (2.15-10.75µM) to the ChT solution showed an increase in the maxima with no spectral changes (Figure 1d).
The fluorescence spectroscopy properties of GaPc1 and ZnPc1 were evaluated for both compounds as fluorescent molecules individually and with the addition of ChT to predict possible interactions (Figure 2a,b).The wide excitation band with a maximum of 280 nm was recorded for the ChT emission of 350 nm (Figure 2a, A).The high-intensity emission maximum of ChT was registered at 350 nm for excitation at 280 nm (Figure 2a,  B).Both fluorescence bands were shown to increase the intensity with the addition of ChT (0.07-2.1 µM) because of the rise of the ChT concentration in the solution.The studies on the fluorescence emission of GaPc1 at an excitation wavelength of 610 nm showed the overlapping of the fluorescence spectra (em: 693 nm) for concentrations over 10.2 µM due to the inner filter effect (not shown).The emission spectra of the mixtures containing both ChT and GaPc1 at the excitation of 280 nm, which is typical for tryptophan (Trp) molecular residues, slightly increased the intensity of the emission band of GaPc1 at 693 nm (Figure 2b).However, at the excitation of 365 nm with the addition of ChT (0.033-0.33 µM), the fluorescence intensity of GaPc1 decreased due to dilution.Following the emission of ChT (347 nm), the fluorescence intensity increased with the splitting of the band in the same solutions.The experiment of ZnPc1 titration with ChT was carried out for the excitation of 280 nm, and the recorded emission spectra of ChT showed an increase in the intensity of the emission band at 347 nm because of the ChT concentration without changes in the band shape.Octa-substituted GaPc1 and ZnPc1 (DMSO) were evaluated and found to have similar fluorescence quantum yields, which were lower than peripheral tetra-substituted complexes with similar substitution groups (0.21 and 0.23).This may be explained through the physical quenching due to the eight peripheral substitution groups to the ring molecule.In addition, the presence of ChT seems to lower the fluorescence of phthalocyanine because of the quenching by the ChT macromolecule.The physical interactions between GaPc1 and ChT were shown by the quenching of GaPc1 fluorescence.The excitation of ChT at 280 nm and also of GaPc1 at 610 nm after the addition of ChT resulted in different spectra.This phenomenon may be explained by the energy transfer between ChT (exc: 280 nm; em: 347 nm), of which the emission may be absorbed by GaPc1 and the fluorescence intensity at 693 nm may increase.The higher concentrations of GaPc1 make changes to the ChT macromolecule, as seen by the emission spectra.

Proteolytic Activity
The proteolytic activity of serine protease ChT is an important property that needs to be purposely investigated [31].Experiments were carried out before in some in vitro studies using casein as a substrate by absorption at 275 nm.In this experiment, it is important to know if the applied ChT is active in a range given by the supplier.Further experiments were carried out for the inhibition of ChT proteolytic activity with the addition of complexes (ZnPc1 and GaPc1) kept in the dark and after irradiation with two light sources:

Proteolytic Activity
The proteolytic activity of serine protease ChT is an important property that needs to be purposely investigated [31].Experiments were carried out before in some in vitro studies using casein as a substrate by absorption at 275 nm.In this experiment, it is important to know if the applied ChT is active in a range given by the supplier.Further experiments were carried out for the inhibition of ChT proteolytic activity with the addition of complexes (ZnPc1 and GaPc1) kept in the dark and after irradiation with two light sources: UV-C (254 nm) and LED 660 nm (Figure 3).The choice of the light source was predicted by the absorption spectra of the target molecules, namely an enzyme (ChT) with absorbance mostly in the deep UV region and phthalocyanine complexes (ZnPc1 and GaPc1) with intensive absorption bands with maximums around 675 nm (LED 660 nm has a spectrum with the maximum of the wavelength in half of the width of the Q-band).

Proteolytic Activity
The proteolytic activity of serine protease ChT is an important property that needs to be purposely investigated [31].Experiments were carried out before in some in vitro studies using casein as a substrate by absorption at 275 nm.In this experiment, it is important to know if the applied ChT is active in a range given by the supplier.Further experiments were carried out for the inhibition of ChT proteolytic activity with the addition of complexes (ZnPc1 and GaPc1) kept in the dark and after irradiation with two light sources: UV-C (254 nm) and LED 660 nm (Figure 3).The choice of the light source was predicted by the absorption spectra of the target molecules, namely an enzyme (ChT) with absorbance mostly in the deep UV region and phthalocyanine complexes (ZnPc1 and GaPc1) with intensive absorption bands with maximums around 675 nm (LED 660 nm has a spectrum with the maximum of the wavelength in half of the width of the Q-band).The inhibition of ChT activity because of photosensitization with complexes (ZnPc1 and GaPc1) was observed.The assay was carried out in parallel for three control experimental groups: (1) only ChT, (2) added complexes (ZnPc1 and GaPc1) in the dark, and (3) ChT irradiated with UV-C or LED 660 nm.The dark controls showed no loss of enzymic activity for the experiments that were carried out in atmospheric oxygen.The red-light exposure (LED 660 nm) showed no influence on inhibition of proteolytic activity.The typical photodynamic inhibition of ChT activity was observed for GaPc1 during irradiation with LED 660 nm (full inhibition).Similar results were obtained for GaPc1 and ZnPc1 in the dark samples.Comparable inhibition was determined for ZnPc1 at LED660 nm and under UVC exposure.The different inhibition ability of complexes (ZnPc1 and GaPc1) refers to the difference in their photosensitization capability.However, both compounds showed a lower inhibition at UV-C irradiation than the control samples with UV-C alone (without phthalocyanine).

Photo-Safety Validation
The photosafety of octa-substituted phthalocyanines (GaPc1 and ZnPc1) and the conjugates with an enzyme (GaPc1+ChT and ZnPc1+ChT) was studied on the model mouse embryo fibroblast normal cell line BALB 3T3.The results are presented by CC 50 values ± SD (µM) at irradiation with a spectrum from a light-emitting diode (LED) known as a solar simulator with a light dose of 10 J/cm 2 (for details, see the Experimental section).The collected data were used to calculate parameters such as the photo-irritation factor (PIF), which for PIF < 2 means the lack of toxicity for GaPc1 and prevalent phototoxicity with PIF > 5 for ZnPc1.Relatively high values of CC 50 , such as 57.74 µM and 58.39 µM, and a factor PIF = 1.023 were obtained for GaPc1, suggesting that it is a very promising compound that is non-cytotoxic under solar light exposure.An increase in the PIF value means the lessening of the toxicity with the addition of ChT to GaPc1 (0.626 vs. 1.023 for GaPc1) and ZnPc1 (1.521 vs. 6.104), which is in advance of the high photosafety of compounds (Table S3 in Supplementary Materials).
The photosafety at solar light exposure for GaPc1 was identical as for the sample of cells kept in the dark (Figure 4).The lack of cytotoxicity was observed for ChT in concentrations below 0.1 mg/mL (not shown).An increase in cytotoxicity on BALB 3T3 cells was determined by the addition of ChT (0.05%), with a big concentration gap between the samples with irradiation and in the dark (Figure 4a).The toxicity of the conjugate ZnPc1+ChT was almost similar to that of ZnPc1 alone (Figure 4b).High phototoxicity was observed for concentrations over 1 µM ZnPc1 and the addition of ChT (0.05%) increase the photosafety.The photosafety of ChT showed no influence on phototoxicity with PIF = 0.726 (Table S3).
The photosafety of octa-substituted phthalocyanines (GaPc1 and ZnPc1) and the conjugates with an enzyme (GaPc1+ChT and ZnPc1+ChT) was studied on the model mouse embryo fibroblast normal cell line BALB 3T3.The results are presented by CC50 values ± SD (µM) at irradiation with a spectrum from a light-emitting diode (LED) known as a solar simulator with a light dose of 10 J/cm 2 (for details, see the Experimental section).The collected data were used to calculate parameters such as the photo-irritation factor (PIF), which for PIF < 2 means the lack of toxicity for GaPc1 and prevalent phototoxicity with PIF > 5 for ZnPc1.Relatively high values of CC50, such as 57.74 µM and 58.39 µM, and a factor PIF = 1.023 were obtained for GaPc1, suggesting that it is a very promising compound that is non-cytotoxic under solar light exposure.An increase in the PIF value means the lessening of the toxicity with the addition of ChT to GaPc1 (0.626 vs. 1.023 for GaPc1) and ZnPc1 (1.521 vs. 6.104), which is in advance of the high photosafety of compounds (Table S3 in Supplementary Materials).
The photosafety at solar light exposure for GaPc1 was identical as for the sample of cells kept in the dark (Figure 4).The lack of cytotoxicity was observed for ChT in concentrations below 0.1 mg/mL (not shown).An increase in cytotoxicity on BALB 3T3 cells was determined by the addition of ChT (0.05%), with a big concentration gap between the samples with irradiation and in the dark (Figure 4a).The toxicity of the conjugate ZnPc1+ChT was almost similar to that of ZnPc1 alone (Figure 4b).High phototoxicity was observed for concentrations over 1 µM ZnPc1 and the addition of ChT (0.05%) increase the photosafety.The photosafety of ChT showed no influence on phototoxicity with PIF = 0.726 (Table S3).The high cytotoxicity ZnPc1 with PIF = 6.104 was observed to be lower with the addition of ChT (PIF = 1.152).The results showed that for concentrations between 0.001 µM and 80 µM, GaPc1 has proper photosafety to solar light exposure (360-1100 nm).In addition, the obtained values suggested the positive low-toxicity effect of a proteolytic serine enzyme ChT as a biomolecule with an advance in photosafety on the cells.

Photodynamic Therapeutic Efficacy
The photodynamic efficacy of octa-substituted phthalocyanine complexes (GaPc1 and ZnPc1) was studied on two skin-originated cell lines, namely a human pigmented melanoma cell line (SH-4) and keratinocyte cell line (HaCaT).The activity of compounds was evaluated after their individual application, as well as for their fresh mixtures with a proteolytic enzyme (ChT).The results are presented as concentration-cell toxicity curves.The dark and phototoxicity of ZnPc1 and GaPc1 were studied on SH-4 versus HaCaT (Figure 5).The toxicity curves for compounds applied individually showed no difference in the inactivation efficiency of both cell lines with better activity for GaPc1 (Figure 5b).The difference in both toxicities and for both cell lines was observed with the addition of ChT for the conjugates GaPc1+ChT and ZnPc1+ChT (Figure 5a,b).The phototoxic effect was higher for GaPc1 than for ZnPc1 in the presence of ChT, with a difference in the effect between both cell lines.The cytotoxicity of both GaPc1 and ZnPc1 was shown to diminish in the presence of a serine protease (ChT).melanoma cell line (SH-4) and keratinocyte cell line (HaCaT).The activity of compounds was evaluated after their individual application, as well as for their fresh mixtures with a proteolytic enzyme (ChT).The results are presented as concentration-cell toxicity curves.The dark and phototoxicity of ZnPc1 and GaPc1 were studied on SH-4 versus HaCaT (Figure 5).The toxicity curves for compounds applied individually showed no difference in the inactivation efficiency of both cell lines with better activity for GaPc1 (Figure 5b).The difference in both toxicities and for both cell lines was observed with the addition of ChT for the conjugates GaPc1+ChT and ZnPc1+ChT (Figure 5a,b).The phototoxic effect was higher for GaPc1 than for ZnPc1 in the presence of ChT, with a difference in the effect between both cell lines.The cytotoxicity of both GaPc1 and ZnPc1 was shown to diminish in the presence of a serine protease (ChT).In vitro PDT was carried out with ZnPc1 or GaPc1 with the enzyme ChT, and the results suggest the advantage of reducing their strong dark and photo-cytotoxicity effects on normal cell lines.This observation is likely due to the competition for the generated reactive species between ChT and the cells' components (lipids, proteins, and other biomolecules).The phototherapeutic index (PI) of the compounds in the presence of ChT is In vitro PDT was carried out with ZnPc1 or GaPc1 with the enzyme ChT, and the results suggest the advantage of reducing their strong dark and photo-cytotoxicity effects on normal cell lines.This observation is likely due to the competition for the generated reactive species between ChT and the cells' components (lipids, proteins, and other biomolecules).The phototherapeutic index (PI) of the compounds in the presence of ChT is presented in Table S4.The phototherapeutic index (PI) was observed to have ratios of 1.3 and 1.27 for the normal cells treated with ZnPc1 or GaPc1 with the addition of ChT, suggesting the lower toxicity of the compounds on normal keratinocyte cells.The index PI was determined, with higher values for tumor cells for GaPc1 (PI: 461 vs. 269 on SH4 cells) and similar values for ZnPc1 (154 vs. 156).

Discussion
Octa-substituted phthalocyanines and gallium and zinc phthalocyanine complexes with eight methylpyridiloxy groups (ZnPc1 and GaPc1) were synthesized by a welldeveloped synthetic procedure [26,27].ZnPc1 was prepared by including an additional step, as shown in Scheme 1.Both complexes were studied and compared for their efficiency in the method of photodynamic therapy (PDT).The water-soluble GaPc1 exists as a monomolecular and photoactive in-phosphate buffer (PB) solution (Figure 1a).The interaction between the proteolytic enzyme α-chymotrypsin (ChT) and ZnPc1 or GaPc1 was assessed using molecular spectroscopy techniques.The changes in the absorption spectra of the resulting mixtures were recorded (Figure 1b,c).The increase in the absorption of the Q-band at 681 nm (GaPc1) indicated a rise in the GaPc1 concentration.It may assume an electrostatic contact between the negative charges of ChT and the complex with eight positive charges (GaPc1 or ZnPc1).This may lead to a high local concentration of molecules, which further creates favorable conditions for the stacking interaction between both molecules may lead to physical conjugates.The spectra of ZnPc1 in buffer solution (PB) are typical for aggregated molecules (Figure 1d).However, the addition of ChT showed only a slight reduction in absorbance due to dilutions without monomerization in the PB solution.The increase in the emission band of ChT and, respectively, tryptophan residue (Trp) fluorescence was recorded for the solutions in buffer, pH 7.8 (Figure 2a).The intensity of the fluorescence of GaPc1 was lessened by an increase in ChT concentration at a constant concentration of GaPc1, as was observed for both excitation wavelengths (exc: 365 and 610 nm).The intrinsic fluorescence of ChT, which has the characteristics of a globular protein containing eight Trp residues, mainly depends on the Trp residue because other amino acids such as phenylalanine (Phe) have a very low quantum yield, and the fluorescence of tyrosine (Tyr) is almost completely quenched by or by an amino or a carboxyl group, or a Trp.However, the presence of GaPc1 in solution was shown to change the emission band (~350 nm) of Trp residue, resulting in a low intensity and the splitting of the emission band (Figure 2b).The fluorescence quenching of the ChT may be explained by the more polar location of Trp residues.One of the attributes of enzymes is their short lifetimes, which are used because of their sensibility to some changes in the media.Farhadian et al. [31] reported variations in the fluorescence intensity of ChT with dynamic quenching during the binding of spermine, a polyamine that is involved in cellular metabolism and is found in all eukaryotic cells.The phenomenon of phthalocyanines quenching enzymic activity was also observed for cholinesterase and amylase, as they are natural physiologically active biomolecules [32,33].
The present study of a photosensitizer (GaPc1 or ZnPc1) and the red LED 660 nm treatment of an enzyme such as ChT showed a significant proteolytic inhibition of its activity.Irradiation with UV-C light was also observed to induce the lower activity of ChT by the Trp residues (Figure 3).The incubation of ChT with phthalocyanine showed a lack of inhibition of the proteolytic activity, which suggests that the compound is not connected to the active center.As is known, the serine proteases widely circulating in the human body have many important functions such as blood coagulation, tissue morphogenesis, cell death, swelling, and wound healing, and assimilation [34].The changes in proteins because of the photosensitization reactions resulted in the degradation of histidine (His), methionine (Met), cysteine (Cys), Trp, and Tyr [35].The photooxidation kinetics of His, Met, and Cys residues were found to be insensitive to the pH changes of media within the range of 6-8, which indicated that the photooxidation rates of proteins must be lower than the corresponding ones for the mixture of the individual amino acids.The sensitized photooxidation of ChT using perinaphthenone, rose bengal, and eosine with a high susceptibility of enzymes to photosensitization in water was reported.
A high photosafety due to the sunlight spectra of exposure was observed for GaPc1 alone and in the presence of ChT on the tested model embryonal cell line BALB 3T3 (Figure 4a).It was determined that there was an increase in toxicity for ZnPc1 used individually, which was much higher for ZnPc1 and ChT (0.05%) for the range of concentrations (Figure 4b).The studies suggested an increase in toxicity due to solar light (LED 350-1100 nm).The phototoxicity to solar light and the calculated indexes PIF < 1 or ~1 suggest high photosafety, except for ZnPc1 (Table S3).
The phototherapeutic effect of GaPc1 and ZnPc1 studied on two cell lines (SH-4 and HaCaT) showed relatively high efficiency for GaPc1 compared to ZnPc1 (Figure 5a,b).The gallium complex has higher activity than the zinc one, which may be related to the ionic radius of the gallium ion and the deformation of the phthalocyanine ring.The cytotoxic effect was lessened, suggesting a lower toxicity because of the addition of ChT.The positive effect of ChT is of importance, especially for the dark toxicity of phthalocyanines (Figure 5a).The phototoxicity of compounds (ZnPc1 and GaPc1) was observed with a slight concentration gap between the curves by the addition of ChT in non-toxic concentrations (Figure 5b).These changes in the photo-and dark toxicity were seen for both cell lines (melanoma tumor and keratinocyte cells).The calculated index (PI) as an indicator of PDT efficiency was seen to increase by a double value for GaPc1 in the presence of ChT (Table S4).
The photosensitizers ZnPc1 and GaPc1 showed differences in anticancer PDT activity on the melanoma cell line (SH-4) as well as with the addition of ChT (Figure 5b).The lowering of the PDT activity due to ChT may be a result of the quenching effect on the generated ROS from the enzyme (ChT) and probably the enzymic inhibition of the tumor mass growth.Thus, it might also influence the diminishment of the harsh toxicity of both photoactive compounds.A difference in the cells' toxicity was observed with the addition of ChT in a PDT study of ZnPc1 and GaPc1.This could possibly be explained by the PAR1 overexpression typical for melanoma cells and probably the inhibition of catalytic activity of the ChT due to photosensitization with GaPc1 or ZnPc1 and LED 660 nm.The option of light screening due to the large molecular structure of ChT may also lead to limitations in the light absorption and lower phototoxicity.
A recent study on pancreatic enzymes showed that they weaken tumor growth by slicing and decomposing the binding proteins of cellular structures [36].The knowledge that the tumor matrix of 3D collagen promotes the migration of tumor cells may be used for protease applications on tumors.In vitro studies in which the tumor microenvironmentassociated structures are not present have also shown antitumor potential of pancreatic enzymes [37].In this study, it was reported that proteases, like trypsin or α-chymotrypsin, cleave extracellular precursors, resulting in the generation of the active form of numerous proteins.For example, trypsin can cleave pro-insulin to generate active insulin through a proteolytic mechanism.The membrane receptors, known as proteinase-activated-receptors (PARs), have been described as potential targets for pancreatic proteolytic enzymes [38].
In the present study, ChT can cleave PAR1, which is overexpressed in many kinds of malignancies, including pigmented melanoma.Chymotrypsin could also make an inhibitory cleavage on PAR1, removing its tethered activating ligand and making the receptor insensitive to the action of other proteinases.The local character of the method plus the positive effect of the proteolytic enzymes such as ChT for anticancer therapy may have several constructive aspects for minimizing the undesirable effects of the PDT procedure.The promising effects were seen after combining the application of a proteolytic enzyme and PDT, then only the PDT.This study presents a promising start for future research on the dual macromolecular assembly of enzymes and photosensitizers in the direction of advanced PDT.

Proteolytic Activity Measurements
The proteolytic activity was determined by an enzyme assay in proteolytic units (CTA) using casein as a substrate.The method was first published by Johnson et al. [39] and further modified [40].This method was chosen because of the specificity in the absorption spectra of the studied phthalocyanines.All assays were made in triplicate and the average of the measurements was taken to evaluate the activity units.One CTA-unit of enzyme caseinolytic activity corresponds to the enzyme amount that releases 1 µeq/min of tyrosin in the substrate (37 • C).The reaction mixtures of the samples consisted of 0.016 mg/mL α-chymotrypsin in 0.06 M Tris buffer, pH 7.4 and either 5 µM GaPc1 or ZnPc1 (control groups).The experimental samples of 2.5 mL volumes were irradiated with a UV-C lamp for 3 min and 15 min by stirring in air-open cuvettes at room temperature (T = 20 • C).The light-emitting diode (LED) 660 nm was applied with the doses (10 J/cm 2 and 50 J/cm 2 ) and did not alter the proteolytic activity of ChT.

Photo-Physicochemical Study
The absorption spectra were measured using a spectrophotometer Perkin Elmer Precisely Lambda 25 UV/Vis with quartz cuvettes with 1.0 cm at room temperature.The stock solutions of GaPc1 and ZnPc1 (~2 mM) in DMSO were used.The dilutions were made with phosphate buffer (PB) with concentration 0.01 M, pH 7.8.The absorption spectra were recorded for the full spectral range 190-750 nm.Absorbance of ChT was observed in the UV-C region with maxima (192 nm and 254 nm in PB (pH 7.8) for different concentrations.The absorption spectra of the mixture GaPc1 and ChT were registered at the titration of a solutions of GaPc1 (3.27 µM or 4.34 µM) or ZnPc1 (4.89 µM) in PB with ChT (5.12-50.6 µM) or the opposite titration, which involved the addition of GaPc1 (2.15-10.75µM) to a solution of ChT (10.1 µM).Fluorescence studies were carried out using Perkin-Elmer LS 55 (Switzerland).The emission spectra were recorded for wavelengths of excitation at 280 nm of tryptophan (ChT) and at 610 nm for phthalocyanines following the previous study [41].

Light Sources
Two different light sources were used in the present studies.Both light emitted diodes (LED) have different spectra and powers of exposure specific to the devices used in the photosafety experiment and the PDT study.The so-called solar simulator has spectra that cover the sunlight.This is a commercially available LED Helios-iO from SERIC Ltd. (Tokyo, Japan).The fluence rate was constant as measured with a power-meter PM 100D with a sensor S120VC (Thorlabs Inc., North Newton, KS, USA).The working dimension between 0.05-50 mW was within the spectrum 360-1100 nm.The intensity was achieved for a distance of 20 cm distance with a normal diffusion in the experimental zone of 1.16 W/m 2 .A second light source is a LED at 660 nm was configured by ELO Ltd. (Bulgaria) with the fixed power density of 100 mW/cm 2 , which was experimentally observed as an optimal fluence rate for in vitro PDT experiments.

Phototoxicity Study
The toxicity studies were performed after serial dilutions of GaPc1 and ZnPc1 from the stocks in DMSO.The incubations were made in the culture medium to the working concentrations between 0.0025-80 µM.The enzyme (ChT) used for the experiment was dissolved in phosphate buffered saline (PBS), pH 7.4, at a concentration of 50 mg/mL used as a stock solution.GaPc1, ZnPc1, and ChT were applied freshly prepared in the culture medium.In vitro tests were carried out on the cells in their exponential stage of grown.After trypsinization the cells were brought to the required cell density in each of the 96-well plates (1 × 10 4 cells/well).The NRU-assay was applied to register the results after treatments.The evaluation of the cells' viability in vitro was carried out.The used experimental protocols followed the known procedure.Briefly, it includes the standard conditions for the cell incubation for 24 h to reach a good adhesion.The cells were incubated after addition of double rise of the testing concentrations for the compound (ZnPc1, GaPc1, ChT and mixtures with ChT).The culture medium was replaced, and the cells were washed before the experiments.The study was performed in parallel plates at the same time.One plate was kept in the dark place to have the dark control in equal conditions as the light exposure for comparative assessment of photo and dark cytotoxicity.The second plate was irradiated with LED 660 nm with light dose of 50 J/cm 2 and 24 h post-irradiation, the medium was replaced with the medium containing the NR dye.Three hours later, the wells were washed with phosphate-buffered saline (PBS, pH 7.4), and a mixture of distilled water/ethanol/acetic acid in a ration 50:49:1 was supplemented.The optical density was measured on a TECAN microplate reader (λ = 570 nm).The cellular toxicity was calculated by Equation (1): Cytotoxicity (%) = {1 − (OD 570 (treated sample)/OD 570 (negative control)} × 100 (1) Other parameters calculated based on the photo-and dark cytotoxicity studies are the phototherapeutic index (PI), toxicity in the dark (IC 50 ), after-irradiation photocytotoxicity (IC 50 ), and photo-irritation factor (PIF), calculated using the Equation (2): PIF = IC 50 (Dark)/IC 50 (LED 360-1100 nm) (2) where Dark is the value in the absence of light and Light is the same after solar LED 360-1100 nm irradiation.Phototherapeutic index (PI) is defined as the ratio of dark to light IC 50 values, and it is used to determine the light-induced effectiveness.The phototherapeutic index was defined as the dark IC 50 value divided by the light IC 50 value and calculated using Equation (3).PI = IC 50 (Dark)/IC 50 (LED 660 nm) (3)

Figure 1 .
Figure 1.Absorption spectra of Zn(II)-and Ga(III)-phthalocyanines (ZnPc1 and GaPc1) in dimethylsulfoxide (DMSO) and phosphate buffer, pH 7.8 (PB) (a); Changes in absorption spectra by addition of chymotrypsin (ChT) with isosbestic points in inset spectra (b) and the same measurements for ZnPc1 (c), and the absorption spectra for the titration of ChT with GaPc1 (d).

Figure 1 .
Figure 1.Absorption spectra of Zn(II)-and Ga(III)-phthalocyanines (ZnPc1 and GaPc1) in dimethylsulfoxide (DMSO) and phosphate buffer, pH 7.8 (PB) (a); Changes in absorption spectra by addition of chymotrypsin (ChT) with isosbestic points in inset spectra (b) and the same measurements for ZnPc1 (c), and the absorption spectra for the titration of ChT with GaPc1 (d).

Figure 5 .
Figure 5. Cytotoxicity of Ga(III)-and Zn(II)-phthalocyanines (GaPc1 and ZnPc1) in the presence of chymotrypsin (ChT), (a) without light (dark toxicity) and (b) at irradiation with a light-emitting diode (LED) at 660 nm with a dose of 50 J/cm 2 (inset: the part of the toxicity curves).p < 0.05 for each point was considered significant.

Figure 5 .
Figure 5. Cytotoxicity of Ga(III)-and Zn(II)-phthalocyanines (GaPc1 and ZnPc1) in the presence of chymotrypsin (ChT), (a) without light (dark toxicity) and (b) at irradiation with a light-emitting diode (LED) at 660 nm with a dose of 50 J/cm 2 (inset: the part the toxicity curves).p < 0.05 for each point was considered significant.