CC BY
47DOI 10.24412/cl-37136-2023-1-39-46
SYNERGIC VASCULAR PHOTODYNAMIC ACTIVITY BY METHYLENE BLUE-CURCUMIN
SUPRAMOLECULAR ASSEMBLY
RODRIGO SILVA1,2, HILDE BUZZÁ3,4, ELI SILVEIRA-ALVES1, KLEBER OLIVEIRA2, VANDERLEI BAGNATO3,5, GUILHERME SOUZA6, LUCIANE ALMEIDA7 AND PABLO GON^ALVES1,8*.
1 Instituto de Química, Universidade Federal de Goiás (UFG), Brazil2Departamento de Química, Universidade Federal de Sao Carlos (UFSCar), Brazil3Instituto de Física de Sao Carlos, Universidade de Sao Paulo, (IFSC, USP), Brazil 4Instituto de Física, Pontificia Universidad Católica de Chile, Chile
5Hagler Fellow, Texas A&M University, College Station, United States 6Instituto de Ciencias Biológicas, Universidade Federal de Goiás (UFG), Brazil
7Universidade Estadual de Goiás (UEG), Campus Anápolis de Ciencias Exatas e Tecnológicas, Brazil 8Instituto de Física, Universidade Federal de Goiás (UFG), Brazil
ABSTRACT
A supramolecular assembly was obtained by combining methylene blue (MB) with a natural plant extract curcumin (Curc) in a stoichiometric ratio of 1:4 in aqueous solution (90% PBS + 10% ethanol) at room temperature. The MB-Curc supramolecular assembly was evidenced by absorption and fluorescence spectroscopies and the stoichiometry and bonding constant were obtained using Cielen's model. Its stability and photostability were evaluated by chromatographic analyzes and UV-Vis absorption. The MB-Curc avoids the aggregation of both isolated compounds and was efficient to produce singlet oxygen (OA = 0.52 ± 0.03). Its potential for photodynamic antiangiogenic treatments was evaluated through vascular effect observed in chicken chorioallantoic membrane (CAM) assay. The results showed intense damage in CAM vascular network by MB-Curc after irradiation, which is higher than the effect of isolated compounds, showing a synergic vascular effect. This combination can be important to avoid cancer revascularization after photodynamic application, and improve this approach's efficacy. The characteristics shown by MB-Curc make it a potential candidate for use in cancer treatments by photodynamic antiangiogenic therapy
INTRODUCTION
The combination of PDT with antiangiogenic therapy has been proposed to avoid cancer revascularization after PDT.[1] Basically, antiangiogenic therapy consists of the use of some drugs to cutting off the blood supply to tumor micro- regions, resulting in hypoxia and necrosis within solid tumor tissues and consequently its death by nutrition starvation.[2] Antiangiogenic therapy also is an alternative for cancer treatment and is effective for some types of cancer, such as metastatic renal cell carcinoma, but not for others, such as breast cancer, melanoma, pancreas and prostate cancers.[3] Another disadvantage of antiangiogenic therapy is the cost, some antiangiogenic drugs commercially available are monoclonal antibodies such as bevacizumab (Avastin®) and DC101, which are very expensive. An alternative to those drugs is the use of natural compounds extracted from plants, which has the advantage of low cost compared to conventional antiangiogenic drugs. Compounds extracted from the rhizomes of the plant Curcuma longa L called curcuminoids, which are composed of curcumin, demethoxy-curcumin and bis-demethoxy-curcumin, are natural products with a wide biological activity, such as antiangiogenics,[4] so on.
We hypothesized that Curc combined with MB could form a supramolecular structure combining the antiangiogenic and photosensitizing properties of Curc and MB. PDT combined with antiangiogenic therapy may result in a synergistic anti-tumor effect that would destroy the tumor and prevent its recurrence. This strategy could be an alternative low cost and efficient for antiangiogenic photodynamic therapy. Herein, we characterized the chemical interaction between these molecules, the formation of a MB-Cur supramolecular complex, evaluated the photo-stability of this complex, and its potential for photodynamic antiangiogenic treatments, which was evaluated through vascular effect caused in chicken chorioallantoic membrane (CAM) assay. We
selected this method because it provides quick results, is cost-effective, simple, has high reproducibility, and presents easy dynamic observation.[5]
RESULTS AND DISCUSSION
Figure 1A shows the effects of Curc presence on the MB absorption spectra. It is possible to observe that the increases of Curc concentration linearly quenches the main absorption band of MB at 664 nm (inset of Fig 1A). Differential UV-Vis spectra were obtained using the solution of MB (4 ^M) as the background of experiments (Fig 1B). In this case, it is possible to see a new redshift band formation at 704 nm for a 1:4 molar ratio between MB and Curc, respectively (inset of Fig 1B). The isosbestic point at 590 nm and the new redshifted band suggests the formation of an MB-Curc complex. On the other hand, the increases of MB concentration (0 - 40 ^M) in Curc solution (4 ^M) did not cause observable changes in the absorption behavior of the Curc band. Possibly, this can be attributed to stoichiometry of the complex. While MB is affected by 4 molecules of curcumin, the curcumin absorption is disturbed by just 25% of this effect (data not shown).
Figure 1: A) UV-Vis absorption spectra for mixtures of MB (4 ¡iM) with Curc (0-40 ¡iM). Inset highlight the decrease of MB absorption band at 665 nm. B) Differential UV-Vis spectra for mixtures of MB (4 ¡iM) with Curc (0-40
¡iM). Inset highlight the band intensity at 704 nm.
Stoichiometry and bonding interaction
The MB-Cur complex formation was evaluated by fluorimetric titration using a multiple binding model developed by Ciele et. al 1998.[6]
log
F-F„
= -logKd + nlog[B]
(3)
F is the fluorescence signal at [B], while Fmin and Fmax denote the fluorescence signal at minimal [B] (absence of B) and maximal [B] (solution is saturated with B in excess), respectively. In a plot of left side of eqn (3) as a function of log [B], the slope provides n, while intersection with abscissa corresponds to logKd. Finally, the binding constant (Kb) can be obtained from a reciprocal relation between Kb and Kd (Kb = 1/ ). MB solution shows a fluorescence emission band centered at 690 nm, which is quenched by curcumin addition (Fig 3A). The absorbance value of the initial MB solution (in absence of Curc) at the excitation wavelength (630 nm) was adjusted to less than 0.1 to avoid the possible contribution of intermediate complexes to the fluorescence intensity. The fitting of the Hill plot (inset of Fig 2A) provides the n = 4.08 and Kb= 3.98x105 M-4, suggesting a stoichiometry 1:4, corresponding to the binding of 4 molecules of Curc to 1 MB (Fig 2B), which is in accordance with data presented in Fig 2.
660 680 700 720 740 760
Wavelength (nm)
(A)
780 800
(B)
Figure 2: A) Fluoresce emission spectra of MB (2 pM) as a function of Cure concentration (from 0 to 50 yM). Inset:
Hill plot. B) Structure proposal of the MB-Curc complex.
Stability of the Curc-MB complex in solution
The variation of the concentration of the species in solution was measured as a function of time to characterize the stability of the Curc-MB complex in solution. MB-Curc complex solution was prepared in PBS (pH 7.4) at 10% of ethanol in a ratio 1:4 of MB (6 ^M) with Curc (24 ^M), respectively. The concentrations were analyzed every 20 minutes for 5 hours to evaluate the stability of these species in solution by HPLC analysis.
The concentration of all species decreased during the experiment (Figure 3A-D). Interestingly, the MB retention time in the MB-Curc complex solution shifted to a shorter retention time during the first 40 minutes of analysis. Subsequently, the retention time of MB in MB-Curc complex solution was similar to the retention time obtained for MB solution, probably due to degradation of the complex in solution. The shorter retention time of MB in the MB-Curc complex solution suggests that the formation of the complex provides a character more hydrophilic to MB.
The concentration of Curc solution reduce of (23.5 ± 0.5) ^M for (11.3 ± 0.5) ^M, degradation of (52.0 ± 1.1)% (Figure 3A) while the concentration of Curc in MB-Curc complex solution reduce of (22.6 ± 0.1) ^M for (14.3 ± 0.1) ^M, reduction of (36.7 ± 0.2)% (Figure 3C). These results demonstrated that the MB-Curc complex has high Curc stability in solution at about 30%. However, the MB stability has low from (10.1 ± 0.1)% in MB solution (Figure 3B) to (22.5 ± 0.7)% in MB-Curc complex solution (Figure 3D), decreasing by 126%, which is about 4 times the increase of stability observed for Curc in MB-Curc complex solution.
The low stability of Curc in buffer systems at neutral-basic conditions has been reported [7]. Studies suggest that the maintaining of the conjugated diene moiety of the Curc, which is observed for both acid pH conditions and metal complexes, may contribute to increasing Curc stability [8]. Thus, our results suggest that the P-ketoenol moieties of Curc could be the site of complexation between Curc and MB.
Omin -20min-40min-1h 1h20 1h40-2h--2h20
2h40-3h-3h20 3h40-4h-4h20-4h40 min-5h
Figure 3: Chromatogram recorded every 20 min for 5 hours for A) Curc, B) methylene blue, C) Curc in the MB-Curc complex solution and D) methylene blue in the MB-Curc complex solution.
MB-Cur complex and aggregation
In solution, the n-stacking interaction between organic molecules of high electronic conjugation can cause the formation of aggregate. Aggregation is a process molecular that affects the properties of monomeric compounds and can reduce their therapeutic activity[9] and then it should be avoided. The formation of the aggregates of Curc was evaluated by the variation of the concentration of ethanol in the PBS buffer using UV-Vis spectroscopy. Thus, solutions of Curc (20 ^M) were prepared in a mixture of PBS buffer and ethanol (10-100%). The data recorded has shown that the decrease of ethanol concentration lesser than 40% induced the appearance of a new blue-shift absorption band of Curc at 370 nm, characteristic of type-H aggregate (Figure 4).
Figure 4: A) UV-Vis spectroscopy of Curc (20 jM) in a mixture of PBS buffer and ethanol (10-100%). B) Relative intensity of new blue-shift absorption band at 370 nm concerning monomer Curc band at 430 nm.
Subsequently, solutions of Cure (20 ^M) and MB (1-10 ^M) in a mixture of PBS buffer and ethanol at 10% were prepared to evaluate the influence of the MB-Curc complex in the trend of formation of type-H aggregate. The data recorded has shown that the increase of MB concentration in solution causes the disappearance of Curc aggregate band, at 370 nm (Figure 5), suggesting that the complex formation decreases the trend of the aggregation of Curc in solution.
300 350 400 450 500 550 0 2 4 6 8 10
A- <nm) [MB] (lM)
Figure 5: A) UV-Vis spectroscopy of Curc (20 jM) and MB (1-10 jM) in a mixture of PBS buffer and
ethanol (10%). B) Relative intensity of new blue-shift absorption band at 370 nm concerning monomer Curc
band at 430 nm.
Photochemical behavior
Singlet oxygen production by MB, Cur and MB-Cur complex was evaluated by the indirect method using uric acid (UA) as a quencher[10]. Figure 7 shows the effects of visible irradiation on the absorption spectra of compounds in the presence of UA, and the emission spectra of LEDs used as the light source. It is possible to see that blue LED irradiation (peak at 450 nm) overlaps the main absorption band of Curc, while red LED (632 nm) overlaps the main band of MB.
The blue LED irradiation practically did not induce any changes in the absorption band of UA as for isolate Curc as MB-Curc complex, indicating no production of singlet oxygen by both samples (Figure 6A and C). Studies have shown that Curc can produce both singlet oxygen and radical species, but when Curc is irradiated by light sources in the continuous mode (CW) a higher singlet oxygen production is observed [11]. However, this production is dependent on the environmental conditions[12] and appropriate formulations are required[13]. In the conditions used in the present work, no reactive oxygen species were produced which explains the negligible UA photodegradation observed.
Photobleaching of Curc was observed in both samples (Curc and complex) when irradiated by a blue LED. However, the photodegradation rate was higher for MB-Curc complex (4.01*10-3 s-1, Figure 6A) than isolate Curc (1.63x10-3 s-1, Figure 6C). On the other hand, MB in the complex kept stable when irradiated with blue LED. Photobleaching of Curc, observed for MB-Curc complex concerning Curc solution, suggests another Curc photodegradation mechanism should be involved such as electron transfer between complex species. Red LED irradiation of MB solution confirms the singlet oxygen production by reduction of the absorption band of UA at 291 nm (Figure 6D). However, for MB-Curc complex, the red irradiation provided a series of photochemical processes in the complex (Figure 6B). In addition to the reduction of the UA absorption band, Curc was photodegraded while the MB absorption band undergoes a slight increase. The increase in the MB band suggests that irradiation could release MB from the MB-Curc complex (Figure 1). Furthermore, the Curc photodegradation constant was about 3.5 times higher (1.41*10-2 s-1, Figure 6B) than obtained using blue LED irradiation (4.01*10-3 s-1, Figure 6A).
MB-Curc presented a of 0.52 ± 0.03, which was the same value found for isolate MB solution (0.52)[14]. These results suggest that the higher Curc photodegradation is not due to an increase of the reactive oxygen species
generation in solution, but is probably due to an electron transfer mechanism between MB and Curc.
Figure 6: UV-Vis spectroscopy data recorded every 10 seconds of irradiation for A) MB-Curc complex irradiated with blue LED, B) MB-Curc complex irradiated with red LED, C) Curc solution irradiated with blue LED, and D) MB solution irradiated with red LED. Normalized emission spectra of ligth sources are presented: a
blue LED (peak at 450 nm) and a red LED (632 nm).
Vascular effect of the methylene blue-Curc complex
The vascular response of CAM was calculated by the ratio between the number of black pixels and total pixels of the processed image normalized by the vascular network immediately before treatment. The control groups, solvent and red light, did not present any effect on blood vessels (data not shown). On the other hand, the other groups showed different vascular effects over time (Figure 7).
120
0.0 0.5 1.0 1.5 2.0 2.5 3,0 3.5 4.0 4,5 5.0
time (h)
H Curc^M Curc+light MB MB+light | MB-Curc ■■ MB-Curc+light
Figure 7: Evolution of vascular effect as a function time of monitoring after 5 min of irradiation for: curcumin in the dark (Curc); curcumin under irradiation (Curc + red light); methylene blue in dark (MB); methylene blue under irradiation (MB + red light); MB- Curc complex in dark (MB-Curc); and MB-Curc complex under irradiation (MB-Curc + red light). The studied concentrations were: [MB] = 100 ¡M, [Curc]
= 400 ¡¡M and for [MB-Curc]: [MB] = 100 jM and [Curc] = 400 ¡¡M, respectively. Regarding the curcumin groups (Curc and Curc+light, Figure 7), CAM vasoconstriction was observed in the dark and after irradiation with red LED. In this case, vasoconstriction cannot be attributed to irradiation, since: (i)
the Cure absorbs in spectral range (320 to 550 nm) different (Fig. 1) of LED emission (570 to 670 nm); (ii) there is no significant difference between the dark and light treatments results, and (iii) no reactive oxygen species were observed when irradiated with red and blue LEDs. Therefore, the intrinsic antiangiogenic activity of curcumin is most likely responsible for the vasoconstriction effect observed in these groups.
Concerning the methylene blue groups (MB and MB+light, Figure 7), it was also observed CAM vasoconstriction in the dark and after irradiation. In the dark, MB causes initial vasodilation followed by a vasoconstrictor effect that reaches a value close to 25% after 5 h of monitoring. This vasoconstriction effect of MB in the dark was previously reported in the literature, and it was attributed to the inhibition of NO synthase enzymatic activity.[15-17] Under irradiation conditions, is observed initially vasodilation (up to 2 hours after irradiation) followed by vasoconstriction (10%) at the end of monitoring (after 5 h). This effect had already been described for Photoditazine® and was attributed to ischemia of superficial blood vessels due to high oxidative stress, resulting in increased blood flow from internal microvessels.[18]
For the MB-Curc complex groups (MB-Curc, MB-Curc+light), the maximum vasoconstriction in the dark was 20%. No statistically significant difference among the vasoconstriction of MB-Curc complex and the other experiments performed in dark (Curc and MB) was observed. After irradiation, the MB-Curc complex group presented a higher reduction of the vascular network (50% of vasoconstriction). This result can be explained based on a synergistic effect between the photodynamic activities of singlet oxygen generated by the MB and the antiangiogenic activity of curcumin. In addition, it was not observed increase of the vascular effect in the first 2 hours of monitoring after irradiation of MB- Curc complex, suggesting that the complex avoided the ischemia of superficial blood vessels induced by the oxidative stress of the PDT.
Curc and MB present different affinities in the cell environment, Curc has a great affinity for the cell membrane due to its high lipophilicity,[19] while MB is positively charged and binds to negatively charged parts of the cells, such as mitochondria.[20] Because of this, the vasoconstrictor effects of these species are performed at different active sites since the curcumin inhibits VEGF and its VEGFR receptors[21] and methylene blue inhibits NO synthase.[15] However, it is probable that it persists in the cellular environment since i) failure to observe the synergistic effect of MB and Curc in the dark and ii) the synergistic effect of photodynamic therapy for MB and antiangiogenic effect of Curc under irradiation. CONCLUSION
Although methylene blue (MB) and curcumin (Curc) have been employed as photosensitizers agents in photodynamic applications, in the present work we obtained a supramolecular structure (MBCurc). The assembly was formed in a molar ratio of 1:4 in an aqueous solution, reduced the aggregation tendency of isolated compounds, increased the stability of curcumin in solution, without affecting the singlet oxygen quantum yield by methylene blue, and presented a high vascular effect. Under irradiation, a vasoconstriction was 50% was observed after 5 h of monitoring. It was attributed to the synergistic effect of the photodynamic activity of MB with the antiangiogenic of curcumin. The excellent vascular response of the MB-Curc makes it a potential candidate for use in cancer treatments by photodynamic antiangiogenic therapy. It is important to note that these compounds are inexpensive, non-toxic, and, in the case of curcumin, a natural product.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge financial support from Funda^ao de Amparo á Pesquisa do Estado de Goiás (FAPEG); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Funda^ao de Amparo á Pesquisa do Estado de Sao Paulo (FAPESP); Coordenado de Aperfei^oamento de Pessoal de Nível Superior (CAPES)
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