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11Porphyrins_ ]V]aKp0r8T8p0LIJ'Jr'J"JÈ>J_Paper
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DOI: 10.6060/mhc200390k
Biocompatible Supramolecular Systems Based on Chlorin e6: Preparation, Photophysical Properties
Inna V. Klimenko,a@ and Anton V. Lobanov3^
aN.M. Emanuel Institute of Biochemical Physics, RAS, 119334 Moscow, Russia
bN.N. Semenov Federal Research Center for Chemical Physics, RAS, 119991 Moscow, Russia
@Corresponding author E-mail: [email protected]
The synthesis methods as well as the data of spectral-fluorescent properties of novel supramolecular systems based on chlorin e6 (CeJ are presented. The effect of various biocompatible excipients such as hydrolyzed polyvinyl alcohol (PVA), poly-N-vinylpyrrolidone (PVP), sodium salt of carboxymethyl cellulose (Na-KMC), dimethylsulfoxide (DMSO), Cremophor® PEG-40 (PEG) on the optical absorption and fluorescence of chlorin e6 is demonstrated. A red shift of the chlorin e6 absorption spectrum when using all presented here excipients is a good prerequisite for increasing tissue permeability for visible light. The fluorescence quantum yield jk of chlorin e6 in systems with all excipients has been calculated. It has been proven, that in all obtained biocompatible systems, except DMSO - Ce6 system, Ce6 molecules disaggregate and charge transfer complexes "excipient - Ce6 " are formed. The high efficiency of such systems as PEG - Ce6, PVP - Ce6 and Na-KMC - Ceforfluorescent diagnosis and photodynamic therapy is noted. The conclusions made in the work can be useful during the new photosensitizer controlled aggregation method development.
Keywords: Chlorin e6, supramolecular systems, spectral characteristics, optical absorption, fluorescence, photodynamic therapy.
Биосовместимые супрамолекулярные системы на основе хлорина е6: получение, фотофизические свойства
И. В. Клименко,a@ А. В. Лобанов3'13
Федеральное государственное бюджетное учреждение науки Институт биохимической физики им. Н.М. Эмануэля Российской академии наук, 119334 Москва, Россия
ъФедеральный исследовательский центр химической физики им. Н.Н. Семенова Российской академии наук, 119991 Москва, Россия
@E-mail: [email protected]
В работе описаны методы синтеза, а также представлены данные изучения спектрально-флуоресцентных свойств новых супрамолекулярных систем на основе хлорина е6 (Хл). Показано влияние различных биосовместимых вспомогательных веществ, таких как поливиниловый спирт гидролизованный (ПВС), поли-N-винилпирролидон (ПВП), натриевая соль карбоксиметилцеллюлозы (Na-КМЦ), диметилсульфоксид (ДМСО), кремофор® ПЭГ-40 (ПЭГ) на оптическое поглощение и флуоресценцию хлорина е. Сдвиг в красную область спектра оптического поглощения хлорина е6 при использовании всех представленных здесь вспомогательных веществ является хорошей предпосылкой для повышения проницаемости ткани для видимого света. Рассчитан квантовый выход флюоресценции jk хлорина е6 в системах со всеми вспомогательными веществами. Доказано, что во всех полученных биосовместимых системах, за исключением системы ДМСО - Хл, молекулы Хл дезагрегируют и образуются комплексы с переносом заряда «вспомогательное вещество - Хл». Отмечена высокая эффективность таких систем, как ПЭГ - Хл, ПВП - Хл и Na-КМЦ - Хл для флуоресцентной диагностики и фотодинамической терапии. Выводы, сделанные в работе, могут быть полезны при разработке нового метода управляемой агрегации фотосенсибилизатора в составе супрамолекулярного комплекса.
Ключевые слова: Хлорин е6, супрамолекулярные системы, спектральные характеристики, оптическое поглощение, флуоресценция, фотодинамическая терапия.
Introduction
Photodynamic therapy (PDT) is now one of the most important treatments for cancer. This method is used in conservative treatment and based on the photosensitizers (PSs, natural or artificially synthesized substances capable of biological tissues photosensitizing) capability to accumulate selectively in tumour or other target tissues and generate singlet oxygen or oxygen-containing free radicals at local exposure of radiation of a certain wavelength corresponding to maximum absorption of PS.[1-6] These active oxygen forms are extremely cytotoxic and play a defining role in malignant cell death. Both in the Russian Federation and in other countries there are a number of drugs of the first to third generations with high antitumor activity for use in clinical practice, which can be conditionally combined into three groups: 1) based on porphyrin: a) derivatives of 5-aminolevulinic acid - precursor of endogenous photosensitizer protoporphyrin ("Alasens"); b) benzoporphyrin derivatives ("Visudine"); c) hematoporphyrin derivatives ("Photophrin", Photogem"); 2) chlorophyll-based PS (chlorins and purpurines ("Photo-lon", "Photoditazin", "Radachlorin", "Foscan"), as well as bacteriochlorins ("Tookad"); 3) dyes (phthalocyanine, naph-thalocyanine ("Photosens", "Thiosens", "Octasens").
But since most marketed PS and drugs do not exhibit the necessary combination of physical, chemical and biological properties for PDT,[7] the evolution of the PDT method is also towards the creation of the so-called "ideal PS" with high selectivity, low phototoxicity and rapid pharmacody-namics. Synthesis of new PS and search for new antitumor drugs are constantly in progress.[7-20] Among the promising ones are chlorin-based compounds, including chlorin e6 (Ce6),[21] as they meet the core requirements for the "ideal PS",[7A22] namely, the existence of the intense absorption band in the long-wave part of the spectrum (600-800 nm), in which biological tissues are more optically transparent (so-called "therapeutic window"[13]); the high capacity to accumulate in the target tissue; the existence of intense fluorescence, which actually provides photodynamic effect, as well as allows to carry out fluorescence diagnostics; the storage stability and virtually no toxicity.[9] At the same time due to the characteristics (X = 660 ± 5 nm,
v max '
e = 41000 mol-1cm-1) chlorin-based compounds show higher depth of light penetration into tissues compared to porphyrins,[71112] and the excitation coefficient of chlorins is 10 times larger than that of the porphyrins.[23]
The availability of raw materials plays a major role in the development of chlorin-based compounds, as chlorins precursor chlorophyll a is produced, for example, from Spir-ulinaplatensis microalgae or nettle leaves.[1112] Chlorophyll, as an element of the usual nutrient media, is not a poison for the humans. Chlorin e6 is synthesized by the anaerobic alkaline hydrolysis of pheophorbide a, formed in turn by treating chlorophyll a with concentrated acids.[11,24] The energy potential of Ce6, as a chlorophyll derivative, is identical to the organism's civilized cells, and the selective accumulation of it in the tumor tissues is due to the capture of Ce6 as a "nutrient medium" for them.
The absence of PS aggregation in solutions, which, in turn, leads to a drop in a quantum yield of singlet oxygen generation, is essential when developing medicines for
PDT. It is known, however, Ce6 molecules form aggregates in water solutions, that considerably reduces their photodynamic activity. The high application potential of Ce6 in supramolecular systems with various excipients was confirmed in our previous works.[1112] Studies of the spectral luminescent properties of Ce6 based supramolecular systems with various excipients such as poly-^-vinylpyrrolidone, polyethyleneglycol PEG-200, bovine serum albumin, chito-san, Triton X-100, sodium hexametaphosphate, polydimeth-yldiallylammonium chloride were conducted. Some recommendations were made for the future use of polyvinylpyrrolidone - Ce6, polyethyleneglycol PEG-200 - Ce6, bovine serum albumin - Ce6 and Triton X-100 - Ce6 systems for diagnostics and therapy. However, a search of more effective chlorin-based dosage forms is still under way. Therefore, we have synthesized new supramolecular compounds based on Ce6 and investigated their photophysical properties.
Experimental
In the present work optical absorption and fluorescence of supramolecular systems based on Ce6 with various excipients, such as polyvinyl alcohol hydrolyzed (PVA), poly-N-vinylpyrrol-idone (PVP), sodium salt of carboxymethylcellulose (Na-KMC), dimethyl sulfoxide (DMSO), Cremophor® PEG-40 (polyethyleneglycol, PEG) have been investigated.
Ce6 kindly provided by the colleagues from Moscow Technological University (Preobrazhensky Department of chemistry and technology of biologically active compounds), PVA ([-CH2CH(OH)-]n, Aldrich, 130 kDa), Na-KMC (food supplement E466, [C6H7O2(C)H)x(CH2COONa)y]n Acros, 250 kDa), DMSO (M = 78.13 g/mol, p = 1.1004 g/cm3), PVP (Sigma, 10 kDa), Cremophor® (hydrogenated castor oil solubilizer, polyethylene-glycol, PEG-40) were used (Figure 1). Purity and identity of Ce6 were confirmed with the MALDI-mass spectrometry method on the Thermo Scientific DSQ II single quadrupole mass spectrometer (Thermo Scientific, USA). The choice of water-soluble excipients (Figure 1) is explained by the fact that they are the most widespread biologically-compatible substances which are the part of pharmacological drugs and cosmetic products. Stock Ce6 solutions with the concentration of 5.0 10-3 mol/L were prepared by dissolution of Ce6 dry sample weight in 20 mL of dimethylformamide (DMF) double-distilled. The received concentration was specified on electron absorption spectrum. For spectral measurements 2 mL of the aqueous solution of one of the excipients and 10 ^ 30 |L of aqua-diluted by 10 times stock Ce6 solution were poured into a quartz 1 cm thick cuvette under stirring. The DMSO, PVP, PVA, PEG, Na-KMC aqueous solutions with the weight concentration of 0.1^1 % were prepared separately. Solutions were stored in the dark at +4 0C.
Absorption spectra (200-900 nm) were recorded with TU-1901 UV-Vis spectrophotometer from Beijing Purkinje General Instruments Co Ltd. Poorly resolved spectra were analyzed by decomposing the spectra into their Gaussian constituents. The fluorescence spectra in the range of 550-800 nm were recorded with Fluorat-02-Panorama spectrofluorimeter (Lumex, Russia). The excitation wavelength was 410 nm. All measurements were conducted at 20 °C. The quantum yield of fluorescence of Ce6 in complexes with excipients was calculated by the relative method using the formula jk = (j^0/sk)/(^4k/s0),[251 where A and A0 are the optical densities of solutions of Ce6 complexed with excipients and of Ce6 aqueous solution, respectively, at a wavelength of 410 nm; I and I0 are the integrated fluorescence intensities (area under the fluorescence spectrum curve) of Ce6 complexed
Макрогетер0циmbl /Macroheterocycles 2020 13(2) 142-146
143
O NOH
Chlorin e
CH2OCH2COONa~ H OH
Na-KMC
-CH—CH2—
J n
PVP
O
II
/S^ H3C CH3
DMSO
OH
PVA
HO-(CH2-CH2-O)n-H
PEG, n=40
Figure 1. Structures of chlorin e6, sodium salt of carboxymethylcellulose (Na-KMC), Cremophor® PEG-40, poly-jV-vinylpyrrolidone (PVP), dimethyl sulfoxide (DMSO), polyvinyl alcohol hydrolyzed (PVA).
n
with excipients and in aqueous solution, respectively. The standard was the free form of Ce6 in an aqueous solution, for which I0 = 0.15[26] (Table 1). The relative error in jk determining did not exceed 7 %.
Results and Discussion
The absorption spectrum of chlorin e6 (Figure 2) contains a specific Soret band (S-band) with the maximum at ~ 404 nm, three peaks of low intensity in the region of 500-600 nm, and, due to the partially hydrated porphyrin rings, a strongly marked peak at I = 653 nm corresponding to the 0-band maximum. Analysis of the spectra in the region of Soret peak shows the same hydrophobicity of all presented systems.[11] We have already said that usually "a blue shift" is formed as a result of strengthening of Ce6 interaction with polar molecules and is connected
0.35
300 400
500
600
700 800
X, nm
Figure 2. Absorption spectra of supramolecular systems based on Ce6: 1. PEG - Ce6, 2. PVP - Ce6, 3. Na-KMC - Ce6, 4. PVA - Ce6, 5. DMSO - Ce 6. Ce6 in aqueous solution.
with chromophore transition to more polar environment, and, in our case, to aqua as a polar solvent.[11] Therefore, the absence of "a blue shift" suggests the identical in size interaction of chlorin e6 molecules with polar aqua molecules, both in the presence of all the excipients used, and simply in the aqueous solution. The spectra of Ce6 solutions with such excipients as PVP, PEG and Na-KMC in the region of Soret band have exhibited the decrease in absorption intensity, and in the case of PEG and Na-KMC a drop of intensity is significant (Table 1). There is also a broadening of the half-width of the absorption maximum by ~ 1-2 nm for the systems with PVP, PEG and Na-KMC, and for the system with PEG there is also a bathochro-mic shift of the absorption maximum by ~ 3 nm. These spectral hypochromic changes in the region of Cope band, most likely, can be explained by the occurring PVP - Ce6, PEG - Ce6 and Na-KMC - Ce6 complexes in solution. Possibly, a chromophore in them is in more ordered form in comparison with its free state in the aqueous solution of Ce6. A shift of the chlorin e6 absorption spectrum to a long-wave region when using all presented here excipients is a good prerequisite for increasing tissue permeability for visible light and reducing the absorption of light by blood hemoglobin in the 500-600 nm region, which plays a significant role in increasing of the PDT efficiency.[311,27] The batho-chromic shift of a 0-band maximum is observed for all presented here supramolecular biocompatible systems. Herewith, the highest shift to the region of long waves is observed for PVP - Ce6 and PEG - Ce6 systems by 12 and 13 nm, respectively. These changes in the absorption spectrum in the presence of excipients are likely related to the solvatochromic effect caused by the presence of a less polar, as compared to an aqueous solution only, environment of a chlorin e That is not to deny a monomerization of the Ce6 aggregated molecules in the presence of excipients. Similar changes in the absorption spectra of the PVP -Ce6 system were also observed in.[28-31] The authors of the work[28] speak about the appearance of the second
D
Table 1. Spectral-fluorecsent properties of supramolecular Ce(¡ based systems.
System ХВ, nm XQ, nm A^/ nm AZq/ nm A410 I nm fl, Д11/2„ nm fl, Is jk
PVP - Ce,. б 404 бб5 24.4 21.3б 0.2б 66S 21.6 33.2 0.19
PEG - Ce б 407 ббб 23.S 1б.99 0.20 б70 19.6 60.2 0.47
Na-KMC - Ce6 б 404 б55 23.2 31.74 0.1б б59 25.0 32.6 0.32
PVA - Ce6 б 404 б54 22.7 21.3б 0.27 658 26.2 28.2 0.16
DMSO - Ce6 б 404 б55 22.7 35.39 0.27 б55 30.4 26.5 0.15
Ce - water б 404 б53 22.7 3б.14 0.27 б57 31.5 26.0 0.15*
peak of low intensity in the region of a 0-band (~ 672 nm) and assign its appearing to the influence of PVP on a mono -merization of Ce6 aggregates. Moreover, with the increasing of PVP concentration in solution the intensity of this peak increases. In our case, there is also a slight multiplicity of the spectrum line of supramolecular systems in the long wave region in the form of low intensity peaks. At the same time the authors of the works[29-30] attribute the hypochromic changes of the PVP - Ce6 systems spectra in the region of a 0-band to the appearance of a complex due to the binding effect from PVP, explaining their point of view by small concentration of chlorin in a system. We believe that in our supramolecular systems both the disaggregation of chlorin e6 molecules and its interaction with excipients with the formation of the charge transfer complexes take place. The appearance of the lines of a porphyrin molecular complex with excipients in the region of the spectra at ~ 600-640 nm is the confirmation of this assumption.
The analysis of the luminescent properties of the presented here systems (Figure 3) has shown that the addition of all excipients leads to the increase in intensity of Ce6 luminescence. The maximum increase in luminescent intensity is specifically attributed to PEG - Ce6, PVP - Ce6, Na-KMC - Ce6 and PVA - Ce6 systems.
It should be noted that the luminescence intensity of the systems is markedly higher when excited with the light
3.0 2.52.01.51.0 0.5 0.0-I
550
600
650
700
750 800
X, nm
Figure 3. Fluorescence spectra of supramolecular systems based on Ce6: 1. PEG - Ce6, 2.PVP - Ce6, 3. Na-KMC - Ce6, 4. PVA -Ce6, 5. DMSO - Ce6, 6. Ce6 in aqueous solution.
of the wavelength at I = 410 nm than when excited with the light of the wavelength at I = 620 nm. It is an indirect indicator of intracomplex energy transfer from excipients to Ce6. The observed strong bathochromic shift of the luminescence maximum, especially in the presence of biocompatible excipients such as Cremophor® PEG-40 and poly-V-vinylpyrrolidone, also indicates the appearance of the excipient - Ce6 complex. It is worth pointing out that chlorins are tetrapyrrole macrocyclic compounds with large areas of ^-conjugation, which are also responsible for the hydrophobicity of the compound. On the other hand, strong intermolecular n-n interactions are responsible for the aggregation of chlorin e6 in aqueous solutions. The excipients presented here, due to the presence of hydrogen bonds, are highly soluble in water. At their addition into a system based on Ce6 there is an increase in its solubility both due to own hydrophobicity, and due to intermolecular interactions between heterocyclic aromatic rings of Ce6 and hydrogen bonds of excipients.
The analysis of the spectral-fluorescent data (Table 1) has shown the increase in the quantum yield of fluorescence value jk for PVP - Ce6 , PEG - Ce6, Na-KMC - Ce6 Tk 6 ' 6' 6,
and PVA - Ce6 systems, which is one more confirmation of consecutive destruction of chlorin e6 aggregates and excipient - Ce6 complex formation. Moreover, the rate of Ce6 aggregates destruction in the aqueous medium is different in the presence of different excipients. It should be noted that spectral-fluorescent parameters of DMSO - Ce6 system remains almost invariable in comparison with similar characteristics of a Ce6 - water system.
Conclusions
Based on all submittes data it is safe to say that application of such biocompatible supramolecular systems as Cremophor® PEG-40 - Ce6, Na-KMC - Ce6 and PVP -Ce6 for PDT and diagnostics is obvious. At the same time solubilization in all solutions of presented excipients except for DMSO prevents aggregation of chlorin e6, ensures its effective stabilization either in monomer fluorescent-active form or in the form of complex with charge transfer.
Acknowledgements. This work was carried out within the state task IB CP (No 01201253304) and the state task of Ministry of science and higher education of Russia (theme 0082-2018-0006, No AAAA-A18-118020890097-1).
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Received 12.03.2020 Accepted 06.07.2020
