Effect of albumin on the fluorescence quantum yield of porphyrin -based agents for fluorescent diagnostics

Accepted Manuscript Title: Effect of albumin on the fluorescence quantum yield of porphyrin -based agents for fluorescent diagnostics Authors: Vladimir V. Zarubaev, Tatyana C. Kris’ko, Elelna V. Kriukova, Tatyana D. Muraviova PII: DOI: Reference:

S1572-1000(17)30286-7 http://dx.doi.org/10.1016/j.pdpdt.2017.09.009 PDPDT 1023

To appear in:

Photodiagnosis and Photodynamic Therapy

Received date: Revised date: Accepted date:

28-4-2017 7-9-2017 17-9-2017

Please cite this article as: Zarubaev Vladimir V, Kris’ko Tatyana C, Kriukova Elelna V, Muraviova Tatyana D.Effect of albumin on the fluorescence quantum yield of porphyrin -based agents for fluorescent diagnostics.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2017.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of albumin on the fluorescence quantum yield of porphyrin -based agents for fluorescent diagnostics Vladimir V. Zarubaev1,*, Tatyana C. Kris’ko2, 3, Elelna V. Kriukova3, Tatyana D. Muraviova2 1

St. Petersburg Pasteur Research Institute of Epidemiology and Microbiology,

197101 St. Petersburg, Russia 2

S.I. Vavilov State Optical Institute, 199053 Saint-Petersburg

3

ITMO University, 197101 Saint-Petersburg, Russia

*

Corresponding author,

Mira str., 14, 197101 St. Petersburg, Russia Tel.+7 911 928 0495 Email: [email protected]

Highlights  Three fluorescence diagnostic agents (FDAs), Photolon, Fotoditazin and Dimegin, are compared for fluorescence quantum yield in PBS and in presence of albumin.  Chlorine-based FDAs possess higher quantum yield in PBS comparing to porphyrin-derived Dimegin.  In presence of albumin, all three FDAs demonstrate similar quantum yield.  The results obtained are important for the practice of fluorescent diagnostics of tumors.

Abstract Background: Among modern methods of diagnostics of tumors, the fluorescent methods are considered ones of the most prospective. Diagnostic agents (DAs) spread throughout the body by the bloodstream, so, the DA molecules are often transported by albumins and can be affected by these proteins. In our study we

evaluate the effect of complex formation between bovine serum albumin (BSA) and three fluorescence DA’s (Photolon, Photoditazin and Dimegin) on their fluorescent quantum yields. Methods: Electron absorption spectroscopy and fluorescence spectroscopy were carried out to calculate fluorescence quantum yields of the DAs using Rhodamine 6G as a standard fluorescent dye. Results: For all three DA’s dissolved in phosphate buffer with pH 7.5 (close to that of blood) the addition of albumin resulted in bathochromic shift of the Soret band as well as change of amplitudes of absorption bands. Similar changes were observed for fluorescence spectra of all DAs that are connected with complex formation between DA and albumin. The presence of isobestic point suggests that DA can present in the solution only in two states, free and BSA-bound. Chlorinebased DA’s demonstrate about 1.5-times higher fluorescence quantum yield in PBS than Dimegin. Nevertheless, the addition of BSA to the solutions of all DA’s decreases sharply their fluorescence quantum yield to approximately equal values. Conclusion: The complex formation between DA and albumin equalize fluorescence efficacies of all studied DAs, so the results of photodymanic diagnostics using the specific DA will depend on other factors.

Keywords Photodynamiс diagnostics; albumin, complex, Photolon; Photoditazin; Dimegin; fluorescence quantum yield

Introduction Among modern methods of diagnostics of tumors, the fluorescent methods are considered ones of the most prospective as they are of high sensitivity, and relatively cheap. Moreover, their use does not require ionizing radiation although allow to conduct continuous real-time monitoring1. They are based on identification of malignant tissue by light irradiation-induced fluorescence of diagnostic agents selectively accumulated within the tumor. Non-invasive fluorescent diagnostics was used successfully, in particular at early stages of

cancer, for monitoring, for instance, of oral tumors 2, skin cancer 3, anal cancer 4, and bladder cancer 5. Porphyrins, the compounds widely presented in biological systems, are often used as reagents for visualization6. Initially, the fluorescence of porphyrin was observed at irradiation of tumor in rat with UV light in 1921. Further, in 1942, the targeted study of hematoporphyrin fluorescence was conducted by injection of the compound that was able to accumulate in both tumor tissue and lymph nodes 1. Later, in order to determine malignant tissues, fluorescent features of porphyrins were evaluated in preclinical and clinical studies. Porphyrins are perfect fluorophores as they are accumulated presumably in malignant tissue and absorb light, mostly, within the “optical window” of living tissue 7. Despite the fact that porphyrins are promising agents for photodetection 6, the clinical impact of porphyrins as a diagnostic agents to localize tumors still requires further development of these molecules to ensure reliable identification of disease. Modern prospective porphyrin-derived diagnostic agents (DAs) for fluorescent diagnostics (PD) and photosensitizers for photodynamic therapy are presented by Photolon8-13, Photoditazin14,15 and Dimegin

16-18

. They possess low

toxicity and high rate of accumulation in pathologic tissues, they also excrete from the body as quickly as after one day. In contrast to Photoditazin and Photolon used in clinical practice, Dimegin is still being at the stage of preclinical trials. In this connection it is less studied. Thus, in the earliest work16 the preferential accumulation of Dimegin within mitochondria was shown, as well as its ability, equally to Photolon19, to induce the apoptotic processes destroying the tumors. Previously17, we studied the absorption spectra of Dimegin in phosphate buffer (рН 7.0), where higher efficacies of singlet oxygen production, luminescence intensity and higher photostability of Dimegin were shown comparing to those for chlorine-based compounds Photoditazine and Radachlorine. The fluorescence spectrum of Dimegin in phosphate buffer and its change after addition of BSA were studied by Dadeko et al. 18

Nevertheless, to the best our knowledge, there are no studies regarding the quantum yield of fluorescence (𝜑𝑥 ) of Dimegin in any medium. The quantum yield is one of the main quantitative characteristics of fluorescence and key parameter for fluorophores comparison 20. Its determination is important for the application of specific DA for fluorescent diagnostics. In addition, of high importance is the medium in which DA is present, as the values of quantum yields strongly depend not only on composition, but also on the form of DA in specific medium, i.e. whether it is mono-or oligomer, bound or not to other components of the medium, etc.

21

. One of accessible model biological

media to study DA’s in vitro is phosphate buffer with pH close to that of blood (aprox. 7.5) with addition of serum albumin at concentration close to that in blood. Albumin contains the most part of plasma proteins (about 60%), and large volume of their molecules makes them the most appropriate transducers of bloodtransporting compounds 22. Thus, after intravenous injection of DA, its molecules are transported by albumins and can be presumably affected by these proteins. Albumin is known to form complexes with porphyrins and chlorines changing their absorbance and fluorescence spectra

28-30

23-27

, thus

. In this connection, it is

important to study the fluorescence quantum yields of DA’s and how they are affected by albumin in phosphate buffer. As far as we know, there are no such data regarding Dimegin and Photoditazin. Previously, we studied the quantum yield of fluorescence of Photoditazin in water that was of 0.05 31. Addition of ovalbumin into the solution decreased the Photoditazin fluorescence quantum yield approximately by one order. Privalov et al.32 showed that Radachlorine, the DA close in properties and composition to Photoditazin, demonstrated similar value of quantum yield of fluorescence (0.04) in three media (ethanol, 0.01 М boronic buffer (pH 9.18) and 0.01 М boronic buffer (pH 7.2) with addition of human serum albumin. The fluorescent properties of Photolon (as well as of Photoditazin and Radachlorine) are determined in high degree by chlorine e6 in their composition. Zhang et al.29 have determined that the value of 𝜑𝑥 for chlorine e6 was 0.05 in aqueous solution

at pH 7–8. In other study33 it was determined as 0.18. According to Isakau et al. 34, the values of fluorescence quantum yields of chlorin e6 in phosphate buffer of pH 6,3-8,5 consisted 0,16-0,18. The formation of the complex of chlorin e6 with polyvinylpyrrolidone being a basis for development of Photolon increased the quantum yield of fluorescence of both components to 0,24 – 0,25. Thus, it can be concluded that the effect of albumin on the quantum yield of fluorescence of Dimegin, Photolon and Photoditazin has been studied rather poorly. The main goal of our study was to evaluate and compare the effect of complex formation between bovine serum albumin (BSA) and three fluorescence DAs (Photolon, Photoditazin and Dimegin) on the DAs fluorescent quantum yield calculated for phosphate buffer with pH 7.5 (close to that of blood). Since Photoditazin and Photolon are chlorine-based drugs and Dimegin is the derivative of deuteroporphyrin IX, based on our results it will also be possible to assess the effect of albumin on different types of porphyrins.

Materials and Methods DAs under investigation: 1. DimeginTM (2,4-di-(alpha-methoxyethyl)-deuteroporphyrin IX disodium salt), lyophilisate, PHARMZASCHITA of FMBA (Russia, Moscow region, Khimki). 2. PhotoditazinTM – concentrate for preparation of infusion solutions, the composition based on N-dimethyl-glucamine salt of chlorine E6; VETA-GRAND (Russia, Moscow) 3.

PhotolonTM

(1,3,5,8-tetramethyl-4-ethyl-2-vinyl-chlorin-6-carbonic-acetic-7-

propion acid sodium-vapor salt, stabilized by PVP), lyophylisate; Belmedpreparaty RUE (Republic of Belarus, Minsk). Photoditazin and Photolon have equal active compound (chlorine E6) and expected to possess, therefore, similar properties. Dimegin is based on deutheroporphyrin and possesses high affinity to tissues and low toxicity (at intravenous application to rats its LD50 is 240 mg/kg) comparing to that of Photoditazin (85 mg/kg) or Photolon (182 mg/kg).

Preparation of solutions. All DAs were dissolved in phosphate-buffered saline (PBS) whose pH is close to that of human blood (7.5). Final concentrations of DAs were based on the requirements for specimens for fluorescent analysis (see below) and were 1.0×10 -5 М and 3.3×10-6 М for Dimegin, 5.0×10-6 М for Photoditazin, and 5.6×10-7 М for Photolon. The same concentrations were used in albumin-containing solutions. For the experiments, lyophilized bovine serum albumin was used of molecular weight of 66 kD, pH 7.2, purity of 98%. Its concentrations in Dimegin-containing solutions covered the range of 7.8×10-6 М to 5.0×10-4 М, and the highest concentration was close to that in human blood. These solutions were tested at wavelength 495 nm. To study the fluorescence at wavelength of 350 nm, lower concentrations of albumin were used. In this case the solutions of Dimegin (3.3×10-6 М), Photoditazin (5.0×10-6 М) and Photolon (5.6×10-7 М) were prepared with 6.3×10-5 М of albumin. Spectroscopy The absorbance spectra of specimens were measured on Shimadzu UV-3600 probe spectrophotometer (Japan), and fluorescence spectra – on spectrofluorimeter Cary Eclipse Varian (USA). To avoid the effects of re-absorption, the concentrations of solutions were chosen in the way when optical density at the wavelength of excitation not to exceed 0.15. All measurements were performed in 10-mm quartz or plastic cuvettes. For calculation of quantum yield of fluorescence of each solution, data of absorbance and fluorescence of standard solution were used. As standard, ethanol solution of Rhodamine 6G with known quantum yield of 0.95 [9 Albert M. Brouwer. Standards for photoluminescence quantum yield measurements in solution (IUPAC Technical Report)/ Pure Appl. Chem., Vol. 83, No. 12, pp. 2213– 2228, 2011] was taken. The quantum yields of solutions under investigation were calculated according to the formula (1) [9 Albert M. Brouwer. Standards for photoluminescence quantum yield measurements in solution (IUPAC Technical Report)/ Pure Appl. Chem., Vol. 83, No. 12, pp. 2213–2228, 2011]:

𝜑𝑥 = 𝜑𝑅6𝐺 ∙

𝑆𝑥 ∙𝐷𝑅6𝐺 𝑆𝑅6𝐺 ∙𝐷𝑥

∙

𝑛𝑥2 2 𝑛𝑒𝑡

, (1)

Where Dx and DR6G are optical densities of experimental DA and standard fluorescent dye, correspondingly, measured at the wavelength of excitation, Sx and SFot – integral intensities (squares under the curves of fluorescence) of experimental DA and standard fluorescent dye, correspondingly, that are proportional to the number of photons of fluorescence, nx, net – refractive indexes of media in which DA and standard due, correspondingly, are dissolved. These indexes have been corrected for BSAcontaining solutions according to Minyaeva et al.35 Results Electron absorption spectroscopy The absorption spectra of all studied DA’s in PBS and BSA-containing solution are presented on Fig.1. As can be seen from the figures, in each spectrum, the Soret band and Q-bands can be clearly identified

36

indicating that DA’s are

completely dissolved and present in there in disaggregated state. For Dimegin, the Soret band is shifted to the left comparing to that for Photolon and Photoditazin. First Q-band of Dimegin is of much lower intensity than that of chlorines (Photolon and Photoditazin) that is connected with hydration of С17=С18 band in the latters 36. When comparing Figs 1A and 1B it can be seen that in the model biological medium, the bathochromic shift of the Soret band was observed for all DA’s. More clearly the shift of Soret band after addition of BSA to Dimegin solution is presented on Fig.2. Moreover, in the case of BSA addition to PBS the amplitudes of DA’s absorbance maximums were changed comparing to PBS-only solutions. These changes were different for different concentrations of DA and BSA as follows from Figs.1 and 2, In addition, isobestic point was detected in the absorption spectra of Dimegin at 1.0×10-5 М (Fig.2). The batochromic shift of Soret band as well as the change of amplitude of maximums of absorption bands

are typical for interaction of porphyrins with albumin that was observed by numerous researchers 18, 24, 28. Fluorescence spectroscopy Two bands were observed in the fluorescence spectra of all solutions (Fig.3). The spectrum of Dimegin was shown to demonstrate strong hypsochromic shift when compared to the spectra of chlorines. This is in agreement with previous results of study of these DA’s in different media – ethanol, phosphate-buffered saline (pH 8.5), and isotonic solution 13, study of Photoditazin in vivo

37

and study

of fluorescence spectrum of Dimegin in distilled water with and without BSA 18. The properties of chlorine-based DA’s appeared highly similar both in terms of absorbance and fluorescence that is explained by similarity of their chemical structure. The fluorescence bands of all DA’s in presence of BSA demonstrated the bathochromic shift comparing to corresponding solutions of the same DA’s in PBS as presented on Fig.4 taking Photolon as an example. The shift is similar to that observed previously, for instance by Ben Dror et al. for porphyrins and chlorins analogs, synthesized with variable numbers of methylenes in their alkyl carboxylic chains 23, by Ol’shevskaya et al. for derivatives of chlorin e(6) and their complexes containing Zn(II), Pd(II) or Sn(IV) 25, and others 18, 29. In the case of BSA addition to PBS, the amplitudes of DA’s fluorescence maximums were changed comparing to PBS-only solutions. These changes were different for different concentrations of DA and BSA (Figs. 3 and 4). Fluorescence quantum yields calculation The calculated values of fluorescence quantum yield of DA’s in the studied solutions are presented in tables 1 and 2. As follows from the Table 1, the chlorinebased DA’s (Photolon and Photoditazin) demonstrate very similar fluorescence quantum yield values which are about 1.5-times higher than that of Dimegin when tested in PBS. Nevertheless, the addition of BSA to the solutions of all DA’s decreases the fluorescence quantum yield to approximately equal values (0.014).

The results in table 2 suggest that the value of quantum yield after BSA addition is not affected by the albumin concentration. Importantly, the quantum yields of fluorescence of Dimegin after BSA addition were of similar values at different concentrations of DA (Tables 1 and 2) despite principal difference between maximums of absorption and fluorescence spectra of these solutions obtained in PBS and model biological medium. Discussion Due to restrictions of equipment, we could not study and compare the quantum yields of fluorescence of all the DA’s under study in phosphate buffer with alumin whose concentration is close to that in the blood. Nevertheless, we succeeded both to study Dimegin at close to blood value (5.0×10-4 М) and low (6.3×10-5 М) concentrations of BSA, in latter case we compared the results with corresponding values of chlorine-based DA’s. These results are important in terms of effect of BSA on the quantum yield of fluorescence. The most important observation is that the addition of BSA to the solutions of all DA’s decreases dramatically the fluorescence quantum yields to approximately equal values, that does not depend on the albumin concentration. It can be suggested that after addition of BSA complexes are formed between DA and BSA that results in frequency shift and changes of amplitudes of maxima of absorption

23-27

. According to previously obtained results, the formation of

complexes was gradual and took about 20 to 30 min. The concentrations of DA’s and albumin as well as their proportion were likely to affect the character of changes of spectra of absorption and fluorescence. Thus, in case of all three DA’s at their low concentration, the addition of albumin resulted in decrease of both absorption at the excitation wavelength and the intensity of fluorescence. In contrast, in case of Dimegin at high concentration, the increase of BSA concentration in the solution led to increase of both optical density and fluorescence intensity. Nevertheless, these changes happened in-phase so they did not change the quantum yield.

The appearance of isobestic point in the spectra of absorption of Dimegin suggests that DA in solution can be only in two states – free and BSA-bound 24, 38, so the phenomenon of lowering the quantum yield of DA’s in model biological medium with BSA can be likely connected with formation of complex between albumin and single molecule of DA, but not with complex between albumin and DA cluster (aggregate). In case of absence of isobestic point in absorbance spectra of DA’s after BSA addition, it is believed that the mechanisms of equilibrium between DA and albumin is more complicated and may involve aggregated structures

24

. These changes were observed for chlorine p6 and pyropheoforbide

following the pH decrease from 7.4 to 5.0: in this case, similar to our observation (pH 7.4), complete disaggregation of DA was achieved, and albumin was bound by tetrapyrrole DA at 1:1 molar ratio, while acidic medium (рН 5.0) prevented the formation of complexes of DA monomers with the protein at studied concentrations range 24, 26. While binding with albumin, porphyrine monomers have an advantage over the aggregates since the high affinity site in human serum albumin structure was identified for them27, 39. The heme-like structures binding site of albumin FA1 is narrow and enough deep pocket on the surface of the subdomain IB, and is formed mainly by hydrophobic amino acids 27, 40. The product of heme oxidation – hemin – is located within this hydrophobic pocket, and its propionic groups interact with basic amino acid residues, lysine, arginine and histidine, near the entry into the pocket. The electrostatic interactions of these groups are undoubtedly important for hemin binding 27. Not only poprphyrins (Dimegin), but also chlorine e6 (the basic structure for Photoditazin and Photolon) is able to bind with serum albumin in a similar manner its three carboxylic groups providing even stronger stabilization by the formation of salt bridges with basic residues 26. Several studies suggest that binding of DA’s with albumin strongly depends on specific side substitutions, nature of the metal and pH of the medium

24-26, 28

. In

this regard, the bulky substituents and decrease of pH weaken the affinity of the molecules to the protein.

Serum albumin is not the only transport protein of the blood. This group is consisted of human serum albumin (blood concentration 35-40 mg/mL), lipoproteins of high (0.35-0.85 mg/mL), low (0.66-1.4 mg/mL) and very low (0.20.4 mg/mL) density, as well as acid alpha-1-glycoprotein

24

. At least one type of

them is required for effective delivery of photosensitizer into tumor 41-43. The main protein competing with HSA in binding to porphyrine-based DA’s are low density lipoproteins (LDP)

44-47

. The nature of complex formation of porhyrins with

lipoproteins differs from that of albumin. In case of albumin, the tetrapyrrole fragment binds to specific site with affinity to heme-like structures while in case of LDPs no such sites have been identified so far, and complex formation goes on due to lipophylic properties of DA’s

48

, LDP being able to bind both monomers and

aggregated forms of DA 24, especially at low concentrations of lipoproteins when higher amount of DA binds to one molecule of LDP thus causing aggregation. The ability of DA to bind specific transport protein listed above is determined in particular by hydrophilic/hydrophobic properties of DA

49, 50

. The lowering of

DA polarity is known to result in increasing of its binding to LDP and, in contrast, decreasing of HSA26. The binding is also affected with the location of charge within the molecule of photosensitizer

45

. Therefore, relatively hydrophilic

preparations bind preferentially with HSA that increases the phototoxicity of photosensitizer for tumor-derived cells, in particular of HepG2 and J774 lines 51. In contrast, lipoproteins serve as transport for hydrophobic compounds, in particular porphyrins and chlorins. To increase their solubility, and therefore making easy their use as DA’s, their structure can be modified in such a way that the compounds become amphiphilic. This guarantees, from one hand, their solubility in water-based solutions and, from another, increases their affinity to plasma membranes of cells and cellular organelles

18, 52

. Thus, to develop the DA’s under

investigation (Photoditazine, Photolon and Dimegin), manufacturers introduced into the porphyrine/chlorine scaffold the additional hydrophilic radicals by either chemical synthesis or by complexation of chlorine with water-soluble polymers

accepted for clinical application. So, at pH 7.5 these DA’s are expected to presumably bind with albumin in monomeric form. The complex formation between DA and albumin results in changing of each component’s properties. In our study, as well as in previous work 53, the reduction of fluorescence quantum yield was observed for porphyrine-based DA’s after binding with albumin. From another hand, however, some authors described the quenching of fluorescence of albumin after contact with porphyrin

54-57

by

mechanisms of ground-state complex formation (main) and electronic energy transfer (additional). The ability of porphyrine-based DA complexed to albumin to produce singlet oxygen is also decreased

15, 58

. Interestingly, the values of quantum yield of

Photoditazin and coproporphyrin-based preparation

15

, differing between each

other almost two-fold in PBS, were equalized to the 0.18 value after addition of albumin that correlates good with our data regarding all DA’s presented in this study. We believe that this can be explained by the fact that all DA’s used in both studies after contact with albumin were in similar microenvironment and bound with albumin by the same binding site. This can be indirectly confirmed by the fact that the dye of non-porphyrin nature, methylene blue15, as reference compound, demonstrated different properties after contact with albumin comparing to porphyrine- and chlorine-based compounds. The absence of concentrationdependence of quantum yield of Dimegin of BSA concentration can be explained by equivalent binding of each monomer of Dimegin with BSA. The mechanisms of effect of BSA on DA’s fluorescence quantum yield should be further studied in additional experiments. Our preliminary study of duration of fluorescence in PBS and PBS+BSA has demonstrated that after addition of albumin the fluorescence duration sharply decreases (data not shown) that correlates with the results of other researchers

29, 53

, who studied chlorine e6

and water-soluble P(V)-porphyrins. In both cases such fluorescence quenching was explained by electron transfer mechanism.

Being complexed with albumin, at pH 7.5, Dimegin has demonstrated similar efficacy in terms of fluorescence quantum yield than clinically approved Photolon and Photoditazin Since the efficacies of fluorescence of all DA’s in presence of albumin are almost equal, the efficacy of application of specific DA for purposes of fluorescent diagnostics should be determined by other factors. Further studies are therefore necessary to identify the most important factors determining the action of prophyrin-based agents for detection of tumors. One of such factors can be characteristics of the equipment that is used for fluorescent diagnostics. Such parameters are important as the excitation wavelength and the curve of sensitivity of detector of fluorescence of tumor. Since modern equipment for fluorescent diagnostics, for instance (LESA-01-BIOSPEC, Russia), or SPECTRUMCLUSTER (Russia) have variable characteristics

59

, it is important to specify the

parameters of equipment tuning for each DA that provide the most effective work. In particular, for Photoditazin, Photolon and other chlorines the fluorescence excitation is done at intensive first Q-band of absorption while for Dimegin the excitation is more effective in green or blue spectral zones in its bands of absorption. The registration of fluorescence of Photoditazin and Photolon provides the optimal efficacy at wavelengths of 625-775 nm while that of Dimegin – at 600725 nm. Also, to increase the efficacy of excitation of DA in vitro, the red shift caused by interaction of DA and blood transport proteins should be taken into account.

Acknowledgements Authors thank A.V. Baranov for providing the access to fluorescent and optical equipment in a framework of the study, and I.V. Martynenko for consulting in the area of methods of fluorescence experiments. The study was supported by the grant of President of Russian Federation for leading scientific schools of Russia (headed by Acad. RAS N.N. Rozanov) and the Government of Russian Federation (grant no. 074-U01).

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Figure legends. Fig.1. – Absorption spectra of DA’s in PBS (a) and in PBS in the presence of 6.3×10-5 М BSA (b): 1- Dimegin (3.3×10-6 М); 2 - Photolon (5.6×10-7 М); 3 Photoditazin (5.0×10-6 М). Fig.2. Absorption spectrum of Dimegin (1.0×10-5 M) in PBS at different concentrations of BSA. Left arrow indicates the isobestic point, right – the tendency of increase of Soret band intensity at with increasing of BSA concentration in solution. Fig.3. Spectra of fluorescence of DA’s in PBS (a) and in PBS in the presence of 6.3×10-5 М BSA (b): 1- Dimegin (3.3×10-6 М); 2 - Photolon (5.6×10-7 М); 3 Photoditazin (5.0×10-6 М). Fig.4. Spectra of fluorescence of Photolon (5.6×10-7 М) dissolved in PBS (1) and in PBS in the presence of 6.3×10-5 М BSA (2). Fluorescence excitation wavelength is 350 nm.

a Figure 1

b Figure 1

Figure 2

а Figure 3

b Figure 3

Figure 4

Table 1. Quantum yields of fluorescence of DA’s in PBS and in the model biological medium (excitation wavelength 350 nm) 𝜑𝑥 (×100) DA

Concentration, M

Dimegin

0,33×10

Photoditazin

0,5×10

Photolon

0,056×10

-5

-5 -5

2,25±0,05

PBS pH 7.5+BSA (6.3×10-5) 1,23±0,02

3,37±0,07

1,44±0,03

3,96±0,08

1,39±0,03

PBS pH 7.5

Table 2. Quantum yields of fluorescence of Dimegin in the solutions with different concentrations of BSA (excitation wavelength 495 nm).

BSA concentration, M 5.0×10 2.5×10 1.3×10 6.3×10 3.1×10 1.6×10 7.8×10 0

-4 -4 -4 -5 -5 -5 -6

𝜑𝑥 (×100) of Dimegin in PBS pH 7.5 +BSA 1,48±0,03 1,58±0,03 1,72±0,03 1,55±0,03 1,58±0,03 1,62±0,03 1,56±0,03 2,40±0,05