Exploring Biological Interactions: A New Pyrazoline as a Versatile Fluorescent Probe for Energy Transfer and Cell Staining Applications

Abstract Fluorescent dyes are used in biological systems, because they are highly sensitive and selective. In this work, we investigated the fluorescent probe properties of 2‐(5‐(pyridin‐2‐yl)‐1H‐pyrazol‐3‐yl) phenol (PYDP) in two media [sodium dodecyl sulfate (SDS) and human serum albumin (HSA)]. Energy transfer parameters, photophysical and thermodynamic parameters of probe were determined. We investigated cytotoxicity of PYDP against colorectal adenocarcinoma cell lines (HT‐29), breast cancer cell lines (MCF‐7) and 3T3‐L1 adipocytes (3T3 L1) cells. The cell staining property of PYDP was monitored using a confocal microscope. The results showed that PYDP binds to HSA, bindings are due to electrostatic/ionic interactions, and the binding process is spontaneous. PYDP was found to exhibit negligible cytotoxicity at high concentrations, and confocal microscope images showed that PYDP stained the cytoplasm of MCF‐7 cells.


Introduction
Fluorescent probes are mostly based on organic molecules.Fluorescence-based techniques are widely used in biological applications due to their simple instrumentation, real-time sensing, high sensitivity, and ease of use. [1]Fluorescence microscopy is essential, especially in live-cell imaging. [2]However, in order to overcome the toxicity difficulties of the fluorescent probe to be used in this technique, the synthesis and discovery of new organic molecules that are easily synthesized, non-toxic and have specific binding properties are important. [3,4]halcones are chromophores [5] and precursors of flavonoids, which are abundant in edible plants. [6]Chalcones (1) or 1,3diphenyl-2E-propene-1-one have an open chain structure with two aromatic rings joined by an α,β-unsaturated carbonyl bridge. [7]They can be easily obtained by Claisen-Schmidt condensation under acidic or basic conditions. [8]The synthesis of chalcone derivatives has attracted attention due to their therapeutic activities such as antitumor, anticancer, antifungal, antidiabetic activities with apparently negligible side effects. [9]halcones are also used to synthesize heterocyclic compounds, which include pyrazolines. [10]Pyrazoline derivatives exhibit blue fluorescence when excited by ultraviolet radiation. [11,12]Therefore, pyrazoline derivatives have been extensively studied for their fluorescence properties and have been used to develop pH probes [13] , to detect Fe 3 + ions [14] , and hydrazine. [15]In addition to their fluorescence properties, pyrazoline derivatives also have high quantum yields, making them ideal candidates for FRET studies. [16]RET is a unique energy transfer process in which energy is transferred from an excited donor fluorophore to a ground state acceptor chromophore. [17]FRET is gaining popularity because it is particularly useful for measuring the molecule structure and molecular interactions, conformational changes and indications of biological events. [18]The effectiveness of FRET depends on 1) the overlap of donor emission spectra and acceptor excitation spectra, 2) the distance between donor and acceptor and 3) the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. [19]he medium used in FRET is critical because the distance and orientation of the fluorophores, which are required for efficient energy transfer, are determined by the medium. [20]ne of the media used in FRET studies is the surfactantbased micelle systems.They have several advantages such as high thermodynamic stability, self-assembly, and the ability interact with both hydrophobic and hydrophilic molecules and resemble the cell membrane structure. [21]Micelle systems which supposed as model membrane systems are usually utilized for the biological applications. [22]Therefore, surfactant systems are often used in FRET phenomenon to study the specific interactions of fluorophores.
Human serum albumin (HSA) is a blood plasma protein that is critical for maintaining osmotic pressure in the circulation. [23]uman serum albumin (HSA) is considered to be an important parameter for defining the health situation of a personal.HSA has two major and structurally important binding sites.Albumin has a hydrophobic pocket in the protein scaffold and can adjust its shape depending on the bound molecule.This property allows albumin to interact with various molecules such as proteins, glycolipids, and drugs. [24]HSA is a significant vehicle in the improving of novel therapeutic agents through its abundant in the circulator system and remarkable acceptor ability. [25]n this paper, we investigated the interaction of a pyrazoline derivative (PYDP) with fluorescent dyes in the micelle system and HSA in water.We also investigated at the cytotoxic effects of PYDP on both cancer cell lines and 3T3-L1 adipocytes performed confocal microscopy imaging of breast cancer cell lines to identify the cell staining characteristics of PYDP.

Spectroscopic properties
Chalcones are bright yellow-colored compounds found in plants and are the precursors of flavonoids. [26]In this study, a chalcone-derived synthetic pyrazoline PYDP, was evaluated for its potential as a biological fluorescent probe.In the first part of the study, we investigated the specific interactions between PYDP and four different chromophores using the FRET technique.The normalized emission spectra of PYDP excited at 350 nm and the normalized absorption spectra of AcO, Fl, PyY and SfT are shown in Figure 1.The result shows that the emission spectrum of PYDP (as a donor) and the absorption spectra of the acceptors overlap, indicating that these pairs are suitable for energy transfer studies.To confirm this, we performed FRET experiments in buffered micelle systems by increasing acceptor concentrations (Figure 2).In micelle systems, the donor molecule is distributed in the micelle scaffold and the acceptor concentration determines the probability of finding acceptor molecules in the vicinity of the donors.Increasing the concentration of acceptors increases FRET  because simply there are more acceptors within the range of energy transfer. [27]We observed that the intensity of the absorption band of the acceptor increases with increasing concentration of the acceptor molecule (AcO), indicating a specific interaction with high affinity between donor and acceptor (Figure 2).If the value of light absorption remains constant when the concentration of a solution is changed, that wavelength is known as the isosbestic point. [28]The isosbestic points were observed at 534 nm and 536 nm for PyY and SfT, respectively, however, no isosbestic points were obtained for AcO and Fl.

Fluorescence quantum yields
The calculation of fluorescence quantum yield is important, because it is used to calculate quenching-rate constants, energy transfer, and radiative and nonradiative rate constants. [30]The fluorescence quantum yield (Φ) is a key property of a fluorophore and indicates the ability of the fluorophore to convert absorbed photons into emitted photons.It is expressed by the following Equation (1): Fluorescence quantum yield data are used to determine the efficiency of the fluorescence process. [30]Two methods can be used to measure the quantum yield: the absolute method and the relative method. [19]The absolute method is applicable in both solid and dissolved phases and is based on a single data collection containing the number of photons emitted by the sample.This method is useful when there is no reliable reference compound that has similar fluorescence characteristics to the compound to be determined.In addition, it requires special equipment.The relative comparison method is the most widely used method and requires a standard reference compound with a known quantum yield value.This method is relatively more accurate since it involves the solution gradient. [19]To calculate the quantum yield of a compound, the integrated fluorescence intensity of the reference compound solution is plotted against the absorbance of the reference compound solution, and the sample with the unknown quantum yield is estimated from this plot.The Parker-Rees method given in Equation ( 2) was used to calculate the quantum yields. [31] where, ϕ s and ϕ r s are the fluorescence quantum yields of the sample and the reference, D s and D r are the areas under the corrected fluorescence spectra of the sample and reference, n s and n r are the refractive index of the sample and reference solvent, OD s and OD r are the optical density measured at the excitation wavelength of the sample and reference, respectively.
A high fluorescence quantum yield is advantageous, because it allows detection of a fluorescence signal even at low concentrations. [32]The fluorescence quantum yields of PYDP with Fl, PyY, AcO and SfT dyes at increasing concentrations in the micellar system (SDS) are listed in Table 1.Quinine sulfate was used as the reference compound in the quantum yield calculation (Φ = 0.58). [33]The fluorescence quantum yield of PYDP was 0.127 (Table 1), which is relatively low as compared to other pyrazoline derivatives reported in the literature.Previously, the quantum yielded of three novel pyrazoline compounds in chloroform were reported to be 0.83, 0.76, and 0.78. [34]Moreover, quantum yields ranging from 0.09 (in water) to 0. 66 (in tetrahydrofuran) were found for the same pyrazoline compound in different solvents.Therefore, the quantum yield of PYDP could change depending on the solvent.
In the fluorescence energy transfer process, the donor's fluorescence quantum yield decreases as the donor transfers its energy to the acceptor.Here, PYDP is the donor molecule.Moreover, the results showed that the quantum yield of PYDP gradually decreased with upon increasing concentration of acceptor dyes (Table 1), which could be attributed to the effective energy transfer from PYDP to the dyes.

Table 1.
Fluorescence quantum yield of PYDP with increasing concentration of acceptor dyes in aqueous SDS solution.

Energy transfer parameters (J and R)
For the fluorescence energy transfer process to occur, the emission spectrum of a fluorophore, called the donor, must overlap with the absorption spectrum of another molecule, called the acceptor.The acceptor does not need to be fluorescent.The donor and acceptor are coupled by a dipoledipole interaction.The extent of energy transfer is determined by the distance between the donor and acceptor, and the extent of spectral overlap.For convenience the spectral overlap is described in terms of the Förster distance (R 0 ).The distance at which FRET is 50 % efficient is called the Förster distance.To quantify the energy transfer efficiency of PYDP as a donor, the spectral overlap integral of the donor-acceptor pair (J) and Förster distance (R 0 ) parameters were calculated.R 0 is given by Equation ( 3): Where k 2 is the spatial orientation factor of the dipole, N is the refractive index of the medium, Φ is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and absorption. [35]he parameter J represents the spectral overlap area of donor emission and acceptor absorption, which can be calculated by the Equation (4): Where F(λ) is the corrected fluorescence intensity at wavelength λ, ɛ(λ) is the molar absorption coefficient of the acceptor at wavelength λ.
The parameters of energy transfer in the micelle system were calculated and the results are shown in Table 2.The overlap of the integrals J for the PYDP and dye(s) pair in SDS ranged from 9.03×10 À 14 to 1.57×10 À 15 .The Förster distance is usually between 2-6 nm. [36]In this study, ranged from 36.85 Å and 40.22 Å for FRET pairs, indicating that energy transfer from PYDP to the dyes is highly likely to occur.FRET efficiency (E) is the fraction of photon energy absorbed by the donor and transmitted to the acceptor. [37]E is based on the distance between the donor and acceptor distance (r) and was calculated using the Equation ( 5): As can be understood, E is increased by shortening the donor-acceptor distance (r).Table 3 shows r and the corresponding E values at different temperatures for all FRET pairs.From this, it can be seen that the PYDP-SfT pair has the shortest distance and the highest E.

Fluorescence quenching mechanism (K sv , k q , K a , E, r(Å), n)
The intensity of fluorescence can be decreased by a wide variety of processes.Such decreases in intensity are called quenching.Quenching can occur by different mechanisms.There are two quenching mechanisms, dynamic and static quenching. [35]Dynamic quenching means that the complex between donor and acceptor occurs during the lifetime of the excited state, whereas static quenching occurs between quenchers and fluorophores in the ground state, and it forms forming a new compound in the ground state. [38]In the static quenching mechanism, the stability of the formed compound decreases with increasing temperature, because the high temperature disassociates weakly formed intermolecular  bonds. [39]We performed a Stern-Volmer analysis to investigate the fluorescence quenching mechanism using the Equation ( 6): Where F 0 and F are the emission intensities of the quencher in the absence and presence of the quencher, respectively.K sv is the Stern-Volmer quenching constant, which indicates the efficiency of quenching.k q is the bimolecular quenching constant, τ 0 is the average lifetime of the fluorophore in absence of quencher and [Q] is the concentration of the quencher. [40]To confirm the quenching mechanism, the double logarithm given by Equation ( 7) was used: where (n) and (K) refer to the number of binding sites and the binding constant, respectively.The plot of F°À F/F versus [Q] at different temperatures are shown in Figure 3.A plot of log(F°À F)/F vs. log[Q] forms a straight line where n is the slope and logK is the intercept (Figure 4).Corresponding results (K sv , K q , K a , E and r (A)) are listed in Table 3.
To recognize between static and dynamic quenching, their different dependence on temperature is examined.Another distinguishing parameter is fluorescence lifetime measurements.Higher temperatures cause faster diffusion and thus larger amounts of collisional quenching.Also, increasing the temperature will lead to dissociation of weakly bonded complexes and therefore less static quenching.Looking at Equation (3) and Table 3, generally the K sv values have been rise with increasing temperature.This indicates dynamic quenching for PYDP-dye(s) donor-acceptor systems.As a matter of fact, FRET is by definition a dynamic quenching mechanism because energy transfer takes place from an excited donor.
The bimolecular quenching constant (k q ) reflects the efficiency of quenching or the accessibility of the fluorophores to the quencher.Diffusion-controlled quenching typically results in values of k q near 1.0×10 10 M À 1 s À 1 .Values of k q smaller than the diffusion-controlled value can result from steric shielding of the fluorophore or a low quenching efficiency.k q is calculated from the equation (K sv = k q .τ0 ) and k q values were below the maximum quenching rate constant (1.0×10 10 M À 1 s À 1 ).The values of n are not consistent with varying temperatures, which could mean that the complex between donor and acceptor is not very stable, and the effect of temperature is minor.The number of binding sites is about 0.5 for PYDP + Fl at 25 °C, which means that one mole of PYDP binds to 0.5 mol of Fl. [40] The efficiency of FRET is based on the distance (r) between donor and acceptor, which should be around 10-100 Å, as previously reported. [41]In this study, the calculated R values range from 40.52 Å to 74.02 Å at different temperatures for all FRET pairs, which is well within the range (Table 3). [42]

Fluorescence lifetime measurement studies
The lifetime (τ) of a fluorophore is the average time between its excitation and return to the ground state.Fluorescence lifetime measurements are used to distinguish between static and dynamic quenching processes.Time-correlated single-photoncounting (TCSPC) technique was used to determine the lifetime decay of PYDP.The emission rates of fluorescence are typically 10 8 s À 1 , so that a typical fluorescence lifetime is near 10 ns (10×10 À 9 s).The nanosecond decay profiles of PYDP with different dyes at 310 excitation nm are shown in Figure 6 and the calculated values are given in Table 4. Wherein τ 1 and τ 2 are the lifetimes, B 1 and B 2 are the fluorescence lifetimes, < τ > is the fluorescence lifetime, χ 2 is the accuracy of the measurement.The fluorescence lifetime values provide information about the interactions of the fluorescence probe with the environment.It should be noted that PYDP has given multiple lifetimes for each system (Figure 5).If there is no acceptor in close proximity to the donor, the donor is partially quenched by the acceptor and a multiexponential decay occurs.Besides, PYDP has shown a longer lifetime in SDS as than in water.This means that there is an interaction between PYDP and negatively charged SDS micelles.Time-resolved donor decays provide a great deal of information about the purity of the sample and the distance between donor and acceptor.Moreover, the fluorescence lifetimes of the PYDP alone (in deionized water) and PYDP with dyes (in SDS) were similar to those of PYDP in SDS solution.In addition, the first lifetimes of the pairs were overall shorter than the second lifetimes, except for the pair PYDP and Fl.This may indicate a complex formation between SDS and Fl, which causes a change in the microenvironment of PYDP.Previously, it was reported that the fluorescence spectra of Fl increased with increasing SDS concentration, suggesting the formation of a complex between Fl and SDS due to hydrophobic interactions. [43]The average fluorescence lifetimes (< τ >) are calculated by taking the mean values of two fluorescence lifetimes by multiplying their percentage values.There are no significant changes in the average lifetime values among the pairs.The value of χ 2 is between 1 and 2, as expected, and the accuracy increases as the value approaches 1.

Thermodynamic parameters and the nature of the binding forces
Next, we calculated the thermodynamic parameters responsible for the binding PYDP with four different dyes using Van't Hoff equation given in Equations ( 8) and ( 9) below. [44] Where R is the universal gas constant, T is the temperature in Kelvin, and K is the binding constant at the corresponding temperature.Plotting lnk against the 1/T plot, the values of ΔH and ΔS are calculated from the slope and cut-off point, respectively.Based on the values of ΔH and ΔS, the nature of interaction could be determined (Table 5).
The results showed that the interaction between PYDP and other fluorescent dyes was stable and spontaneously driven by negative ΔG 0 values (Table 6).All donor-acceptor pairs exhib-ited negative ΔH and positive ΔS values, suggesting that all interactions were due to the electrostatic and ionic interactions.

HSA studies
So far, we have discussed the suitability of PYDP as a fluorescent donor probe for biological systems.In this section, we have studied the interaction between HSA (donor) and PYDP (acceptor) in the aqueous environment.HSA have been used as a model protein for many and diverse biophysical and physicochemical studies.The intrinsic fluorescence feature of HSA is due to two types of amino acids: one is tryptophan in subdomain IIA (Trp-214) and the other is 18 tyrosine residues. [45]hen a compound interacts with HSA, the fluorescence property of HSA decreases depending on the proximity of the compound to these amino acid residues.

Absorption and fluorescence spectral properties
The fluorescence spectra of HSA (1.0×10 À 6 M) and the absorption spectra of PYDP are shown in Figure 6.Upon excitation at 290 nm, the fluorescence spectra of HSA showed an emission maximum at 345 nm and the absorption spectra of PYDP showed maximum at 312 nm.We also measured the fluorescence spectra of HSA alone and in the presence of an increasing concentration of PYDP from 3.3×10 À 5 to 2.7×10 À 4 M.The results showed that the fluorescence intensity of HSA decreased significantly with increasing concentration of PYDP with a shift to high wavelengths, suggesting that there is an interaction between HSA and PYDP due to a non-radiative

Fluorescence quantum yields
One of the parameters used to quantify protein interactions in vivo and in vitro is quantum yield. [46]As expected, the quantum yield of the donor should decrease when the acceptor is close to the donor for energy transfer to occur.We observed that the quantum yield of HSA substantially reduced with increasing concentrations of PYDP (Table 7), which is in the agreement with the result of spectral data.

Energy transfer parameters (J and R)
As shown in Figure 6, HSA has a broad fluorescence spectrum peak and overlaps very well with the absorption spectra of PYDP.Using these curves and quantum yields for water solutions, J and R were calculated.The distance is important for efficient energy transfer from donor to acceptor.Förster radius (R) was calculated as 29 Å for HSA-PYDP pair, indicating that donor and acceptor are near, and FRET occurred with high probability.The value of overlap integral (J) for HSA -PYDP pair was found as 7.94×10 À 13 M À 1 cm À 1 nm 4 .

Fluorescence quenching mechanism (K sv , k q , K a , E, r(Å), n)
To determine the quenching mechanism, we used the Stern-Volmer equation.The plots of F°À F/F vs.
[Q] and log((F°À F))/F vs. log[Q] are given in Figures 7A and 7B, respectively.In the HSA-PYDP pair, K sv values were decreased with increasing temperature (from 25 °C to 45 °C).K q is calculated from the Equation ( 6) and the fluorescence lifetime τ 0 of HSA was reported approximately as 10 À 8 s. [47,48] The k q value is higher than quenching rate constant (1.0×10 10 M À 1 s À 1 ), which indicates a diffusion-controlled quenching mechanism. [49]k q values that appear larger than the diffusion-controlled limit generally demonstrate some kind of binding interaction.The number of binding sites and the distance between the PYDP and HSA were similar at all temperatures (Table 8).The fact that the n values are close to 1 regardless of the temperature indicates that the interaction of HSA and PYDP is from a single region.It also means that the temperature increase does not damage the Table 7.The fluorescence quantum yield of HSA with increasing concentration of PYDP in aqueous media.
Temperature HSA-PYDP interaction.FRET efficiency (E) depends on the distance of donor and acceptor, and the calculated E values were similar at different temperatures due to the comparable donor-acceptor distance (Table 8).

Fluorescence lifetime measurement studies
The

Thermodynamic parameters and the nature of the binding forces
The type and the magnitude of the binding forced are mainly determined by thermodynamic parameters. [35]The Van't Hoff plot was used to determine the parameters including ΔH, ΔS and ΔG were estimated.ΔH 0 and ΔS 0 are found as À 12.02 and 0.06, respectively.As the binding is enthalpically and entropically favorable (ΔH < 0 and ΔS > 0), it is more likely that the binding between HSA and PYDP occurs due to electrostatic/ ionic interactions.We also found the value of ΔG 0 to be À 28.67, indicating that the binding occurs spontaneously.

Cytotoxicity
[52] As can be seen from Figure 9 show that PYDP has no cytotoxic effect on cancer and healthy cell lines at a concentration of 100 μM.On the other hand, at higher concentrations (250 μM), it still has no significant effect on cancer cells, while it has little cytotoxic effect on healthy cells.

Confocal Microscope Examination
From the application point of view, we investigated whether PYDP molecule could be used as a fluorescent dye to stain the fixed cell.onics; Red fluorescence: images stained with verteporfin have been given.Confocal microscopy images of MCF-7 cell lines showed a cytoplasmatic distribution for both Verteporfin (red signal) and rhodaminated-micelles (yellow signal).Besides, it is well known that preferential site of action of Verteporfin is the mitochondria.Here, pluronic micelles were used to photosensitizer Verteporfin. [53]The images (Figures 10B and 10 C) show that PYDP penetrates the fixed cell membrane and exhibits fluorescence properties in both the cytoplasm and intracellular compartments.This suggest that the PYDP molecule could be used as a cytopainter.It is possible that the amine-reactive group in PYDP binds covalently to cytoplasmic proteins, probably throughout the endoplasmic reticulum, allowing uniform cell imaging. [54]It is clear from the images that PYDP does not stain the nucleus, which could mean that PYDP does not fluorescence in the presence of DNA.

Conclusions
The document provides a detailed analysis of the interaction of PYDP with different dyes and HSA in aqueous media.PYDP was synthesized and characterized (by FTIR, 1 H NMR, and 13 C NMR spectroscopy), and investigated for its potential as a fluorescent probe using FRET based methods.Energy transfer parameters, quantum yields, quenching mechanisms as well as lifetime measurements have shown that PYDP acts as a donor for the dyes Safranin T, Acridine O, Pyronin Y, and Fluorescein dyes in SDS media and as an acceptor in HSA due to the overlapping spectra and the proximity between the molecules.The study involved the calculation of thermodynamic parameters.The results reveal that the interaction between PYDP and other fluorescent dyes was stable and spontaneously driven by negative ΔG 0 values.Additionally, all donor-acceptor pairs exhibited negative ΔH and positive ΔS values, indicating that all interactions were due to the electrostatic and ionic interactions.We also examined the cytotoxic effects of PYDP against MCF-7 cell lines as well as the corresponding 3T3-L1 cell lines using the XTT assay.This showed that PYDP has no cytotoxic effects on cancer and healthy cell lines at a concentration of 100 μM.However, at a higher concentration of 250 μM, it still has no significant effect on cancer cells, while it has a little cytotoxic effect on healthy cells.Finally, the cell staining property of PYDP was monitored using a confocal microscope.The results showed that PYDP penetrates the fixed cell membrane, exhibits fluorescence properties, and it selectively stained cytoplasm.This suggested that the PYDP molecule could be used as a cytopainter.Overall, the study provides valuable insights into the interaction of PYDP with different dyes and HSA in aqueous media.The findings of this study could have significant implications in the field of biophysical and physicochemical research and could pave the way for the development of novel fluorescent probes and cytopainters.

Experimental Section
General procedure for the synthesis of chalcone derivative 2-(5-(pyridin-2-yl)-1H-pyrazol-3-yl) phenol (PYDP) PYDP was selected to investigate its potential as a fluorescent probe.The molecular structure and the synthesis of PYDP is described in Scheme 1. Chalcone derivative was synthesized by the reaction of 2-hydroxyacetophenone with 2-pyridinecarboxaldehyde in a yield of 67 %.Then, the reaction mixture was stirred in a microwave oven (800 watt) at 70 °C for 4 min using hydrazine hydrate to obtain the final product. [28]Characterization of PYDP was carried out using FTIR, 1 H NMR, and 13 C NMR spectroscopy.

Figure 2 .
Figure 2. Fluorescence energy transfer spectra of PYDP in the presence of various concentrations of different acceptor dyes: (A) AcO, (B) Fl, (C) PyY and (D) SfT in SDS media.

Figure 5 .
Figure 5. Lifetime decay profiles of PYDP determined at an excitation wavelength of 310 nm and an emission wavelength range of 460-475 nm in aqueous buffered solution and micelle.
fluorescence lifetime of HSA in the absence and in the presence of PYDP are shown in Figure 8.The fluorescence lifetime of HSA reduces in the presence of PYDP indicating the occurrence of energy transfer from the donor to the acceptor.The fluorescence lifetime profile of PYDP in aqueous media.The shorter (τ 1 ) and the longer (τ 2 ) fluorescence lifetime values of HSA were obtained as 1.618 ns and 4.300 ns, respectively.Since B 2 (84.34 %) is greater than B 1 (15.66 %), it is clear that the longer lifetimes are more apparent in the PYDP + HSA system (χ 2 : 1.403).

Figure 7 .
Figure 7. Stern-Volmer plot and logarithmic Stern-Volmer plot of HSA binding with PYDP in aqueous media.
, PYDP at 100 μM (p > 0.05) did not show a statistically significant change in the proliferation of MCF-7 cells compared to cells in the control group.In contrast, a 12 % inhibition (*p < 0.05) was observed for the HT-29 cell line when the PYDP concentration was increased to 250 μM.The compound caused an increase in cell proliferation at 100 μM compared to the control group (*p < 0.05), while at 250 μM (p > 0.05) it caused not a statistically significant change on HT-29.In the healthy cell line 3T3-L1, PYDP caused no significant change in cell proliferation at 100 μM (p > 0.05), but it caused statistically significant inhibition at 250 μM (**p < 0.01).All these data

Table 3 .
Energy transfer parameters, quenching parameters and binding constant of PYDP in SDS environment.

Table 4 .
Fluorescence lifetime values of PYDP in different environments.

Table 5 .
Thermodynamical parameters of molecular interactions.

Table 6 .
Thermodynamic parameter values for the interaction of PYDP with different fluorescence dyes.