Suppressing singlet oxygen formation from 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin using polyion complex micelles

Abstract

Suppressing the overproduction of harmful active oxygen species is very important. We report that the production of ¹O₂ from TPPS can be reduced upon the formation of polyion micelles with PMVP₄₁-b-PEO₂₀₅. The amount of ¹O₂ can be controlled successfully, which affords a new thinking of disease treatment and oxidation resistance of cells.

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Polyion complex (PIC) micelles have attracted increasing attention in the field of macromolecular self-assembly, since they are found to be very useful in a wide variety of fields such as controlled release,¹ transduction of genes,² sensors,³ nanoreactors⁴ and delivery of biomolecules.⁵ Different from surfactant micelles with a simple hydrophobic core covered by hydrophilic head groups, the PIC micelles often have core–shell structures; thus, the micellar core is deeply buried in the forest of the corona.⁶ This makes it difficult for small molecules to defuse into the micellar core, and the component that is enriched in the micellar core, such as DNA and proteins, are well protected.⁷

Moreover, the packing of small molecules such as porphyrin derivatives in the core of PIC micelles is also of interest since porphyrins play key roles in a wide variety of biochemical processes,⁸ electronic devices⁹ and biomimic chemistry.¹⁰ For instance, Shi et al. studied the aggregation and optical performance of the dye 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) in acidic aqueous media. They found that TPPS still retains the ability to form pH dependent H and J aggregates in the core of the PIC micelles formed with PEO–P4VP.¹¹ Considering that various porphyrin derivatives are relevant to many biochemical processes, especially in producing singlet oxygen (¹O₂),¹² it will be very interesting to study the effect of PIC micellization on the ability of producing ¹O₂, preferably, in neutral aqueous solution, which is closer to the physiological pH of living organisms.¹³ Usually, ¹O₂ is continuously produced in the oxygen metabolism of living organisms, and controlling the quantity of ¹O₂ at a normal level is very crucial for signal transduction, immune system control, cellular signalling, blood pressure modulation and metabolism.¹⁴ When overproduced or the levels of antioxidants become seriously depleted, the high quantity of ¹O₂ may cause oxidative stress through the oxidation of biomolecules, which can irreversibly damage the lipids of cellular membranes, proteins, enzymes, carbon hydrates or DNA. This may result in diseases and aging.¹⁵ For instance, porphyria, one of the disorders of porphyrin metabolism in organisms, is triggered by porphyrin accumulation, which overproduces ¹O₂ via irradiation, leading to a series of harmful effects.

On the other hand, as a major bactericide, ¹O₂ has significance in photodynamic therapy (PDT) to destroy tumors.¹⁶ In this regard, many efforts have been made to enhance the amount of ¹O₂ to make full use of the promising noninvasive cancer treatment approach of PDT.^17,18 Till date, there are no reports on how to reduce the amount of ¹O₂ to avoid the harm of over expression on cells. Herein, we show that the production of ¹O₂ can be effectively decreased upon PIC micelle formation. This indicates that upon the formation of PIC micelles, we can effectively tune the production of ¹O₂ in a negative way, which may offer an important approach to suppress the harmful accumulation of ¹O₂.

In this study, we first show the formation of PIC micelles with TPPS and PMVP₄₁-b-PEO₂₀₅ block copolymer in neutral aqueous solution, and then report the effect of PIC micelles on the production of ¹O₂. The ability of the antioxidant action and photostability of PIC micelles endows them with potential applications in the treatment of diseases triggered by the harmful accumulation of ¹O₂. Finally, we show that the production of ¹O₂ can be controlled by tuning the intactness of the PIC micelles with the addition of NaCl to shield the charge interactions. We believe that a controllable ¹O₂ generation would afford the base and a new thinking of PDT.

In neutral aqueous solution, TPPS molecules are tetra-anionic and exist in the form of a monomeric free base (H₂TPPS⁴⁻) due to electrostatic repulsion between H₂TPPS⁴⁻.¹⁹ Upon mixing with the positively charged double hydrophilic block copolymer PMVP₄₁-b-PEO₂₀₅, a strong Tyndall effect was observed (inset in Fig. 1), suggesting the formation of polyion complex micelles, which was confirmed later with TEM observations (Fig. 2).

Fig. 1 UV-vis absorption spectra of the TPPS and PIC micelles in neutral aqueous solution.

Fig. 2 TEM images of PIC micelles. (a) and (c) are the micelles without and with 0.3 M NaCl, respectively; (b) FF-TEM micrograph of micelles without NaCl. The concentration of TPPS and the charged unit MVP in PMVP₄₁-b-PEO₂₀₅ is 20 and 80 μM, respectively, to reach charge balance between the negative and positive charges.

The formation of micelles leads to a significant change in the UV-vis absorption of TPPS. The black line in Fig. 1 displays the UV-vis spectrum of TPPS in a neutral aqueous solution. The characteristic features of an intense Soret band at 413 nm and four weak Q bands at 515, 553, 580 and 634 nm are observed. Upon the formation of micelles, the Soret band of TPPS was significantly reduced by a ratio of ca. 30%. Moreover, the Q bands were shifted to 517, 554, 584 and 647 nm. These results suggest that the formation of PIC micelles have changed the local environment of TPPS, but the TPPS molecules in the micellar core still exist in the form of monomers. Namely, no H or J aggregates of TPPS are formed in the micelles, which is different from the behaviours in acidic solutions.²⁰

The formation of micelles also results in the disappearance of the proton signals of TPPS in the ¹H NMR spectrum. Fig. S1† shows that before interaction with PMVP₄₁-b-PEO₂₀₅, the typical protons of TPPS are discernible, but signals for these protons were silenced in the presence of PMVP₄₁-b-PEO₂₀₅, suggesting the TPPS molecules have been packed into the micellar core.¹¹ TEM observation indicates the average diameter of the micellar core is about 12 nm (Fig. 2a), which is in accordance with other PIC micelles formed with PMVP₄₁-b-PEO₂₀₅. Unfortunately, the overall hydrodynamic radius of the micelles can hardly be obtained with dynamic light scattering (DLS) due to the strong absorbance of 500–650 nm laser by TPPS in neutral solution. However, the thickness of the protecting PEO corona can be estimated from the TEM images by measuring the closest distance between two neighbouring micellar cores. This value is about 15–20 nm, which is also in good agreement with our previous report.^21,22 Furthermore, the overall structures of the PIC micelles formed with TPPS and PMVP₄₁-b-PEO₂₀₅ are fluid-like; thus, an irregular morphology was obtained when the sample was subjected to free-fractured TEM observations (Fig. 2b). The average size for the micelles obtained in FF-TEM is about 40–50 nm, which is in line with previous estimation.

Porphyrins are well-known for their ability to generate ¹O₂, which is caused by the transference of energy to triplet oxygen from triplet state porphyrin photosensitizer induced by light. To evaluate the activity of the photosensitizer of TPPS, we employed the “iodide method” to detect the formation of ¹O₂. The principle behind this method is that the amount of I³⁻ produced by oxidation of I⁻ with ¹O₂ is proportional to the concentration of ¹O₂ under continuous irradiation.²³ Therefore, the UV absorption of I³⁻ can be used to track the production of ¹O₂.

Therefore, we recorded the UV spectra changes of TPPS in the presence of KI before and after the PIC micelle formation (Fig. 3), under continuous UV irradiation. The bands at λ = 353 nm and λ = 287 nm are the characteristics of absorption from tri-iodide. Fig. 3a shows that these bands keep increasing with the prolongation of the UV irradiation time, suggesting the accumulation of ¹O₂ in the free TPPS solution. It is evident that these bands get much lower in the PIC micelles than those of free TPPS. Hence, the formation of PIC micelles indeed suppressed the production of ¹O₂ (Fig. S3b†). In Fig. 4, we compared the absorption at λ = 353 nm in different solution environment. It is found that the absorption was reduced by a ratio of about 30% in the micelles. Since the absorption of tri-iodide is proportional to the production of ¹O₂, this indicates that PIC micelles can suppress the ¹O₂ formation by a factor of about 30%. This value is rather significant in the terms of maintaining the level of ¹O₂ at a normal level. This means that the lower efficiency of producing ¹O₂ is indeed realized by entrapping the porphyrins into the core of PIC micelles. We expect that this decreases is the possibility of energy transformation from porphyrin to oxygen molecules to produce ¹O₂.²⁴

Fig. 3 UV-vis absorption spectra in dependence on irradiation time for (a) TPPS and (b) the PIC micelles; both in iodide solution with c(iodide) = in 0.05 M.

Fig. 4 (a) UV-vis absorption spectra of TPPS and the PIC micelles in dependence on NaCl concentration (b) iodide absorption at λ = 353 nm of TPPS and the PIC micelles in dependence on NaCl concentration; both in iodide solution with c(iodide) = in 0.1 M and for 10 min irradiation time.

In order to gain more physical insight about the reduced production of ¹O₂, NaCl was added to the PIC micellar system to disintegrate the micelles. It is well known that the electrostatic complex micelles, driven by electrostatic interaction, are sensitive to the ionic strength. Furthermore, the increase in the ionic strength of the solution could diminish the driving force for micellization by shielding the electrostatic interactions between TPPS and PMVP₄₁-b-PEO₂₀₅.²⁵ It was found that with increasing the salt concentration, the absorption indeed partly recovered, suggesting that the disassembly of micelles helps the production of ¹O₂. We noticed that the level of ¹O₂ never completely resumed to that in the free TPPS system. Even at NaCl concentration as high as 0.7 M (Fig. 4a), where most the PIC micelles are broken (Fig. S2b†), the production of ¹O₂ is still much lower than that in the free TPPS system. Further increase in the concentration of NaCl, again resulted in a decrease in the absorption (Fig. 4b). It should be noted that the addition of appropriate NaCl did not change the characteristic UV absorbance of TPPS (Fig. 4a and S4†), and the micelles were completely broken in the presence of >0.7 M NaCl (Fig. S2c†). Control experiments revealed that the presence of NaCl may decrease the production of ¹O₂, too. However, the ability of small ions in this regard is very limited, which only becomes significant at salt concentration beyond 0.7 M. Especially, in practical applications, the ionic strength in biological environment corresponds only to about 0.15 M NaCl. At this low salt concentration, the suppression of ¹O₂ by NaCl is negligible, and the contribution of PIC micelles is dominant.

Conclusions

In conclusion, the approach of polyion complex (PIC) micelles is verified effective in suppressing the overproduction of ¹O₂ from anionic 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS). The production of ¹O₂ can be reduced by a factor of 30% via the formation of the PIC micelles. This factor is very significant in keeping the level of ¹O₂ at a normal level. We believe that this study would be helpful in understanding the characteristics of bio-aggregates and the practical treatment of diseases triggered by harmful accumulation of ¹O₂.

Acknowledgements

This study is supported by the Major State Basic Research Development Program of China (973 Program, 2013CB933800).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16259e

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