Effect of aggregation behavior on linear and nonlinear photophysical properties of lipid-water amphiphilic photosensitizers

Effect of aggregation behavior on linear and nonlinear photophysical properties of lipid-water amphiphilic photosensitizers

Journal of Photochemistry & Photobiology A: Chemistry 372 (2019) 206–211 Contents lists available at ScienceDirect Journal of Photochemistry & Photo...

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Journal of Photochemistry & Photobiology A: Chemistry 372 (2019) 206–211

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem

Effect of aggregation behavior on linear and nonlinear photophysical properties of lipid-water amphiphilic photosensitizers Xiaopu Wanga,b, Xing Huanga,b, Yuxia Zhaoa, a b

T



Technical Institute of Physics and Chemistry, CAS, Beijing, 100190, China University of Chinese Academy of Sciences, Beijing, 100049, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Photosensitizers Lipid-water amphipathy Aggregation behavior Two-photon fluorescence Polyethylene glycol

With the wide application of two-photon technology in biological field, lipid-water amphiphilic chromophores containing a large conjugation structure are required. In this study, six polyethylene glycol (PEG) modified benzylidene cycloalkanone photosensitizers (PSs) were selected as models to investigate the effect of their aggregation behaviors on their photophysical properties in deionized water. The results showed that all of these PSs could form aggregates at a suitable concentration in aqueous solution. However, the aggregation behaviors of symmetrically modified PSs (B2, X2 and M2) and asymmetrically modified ones (B3, X3 and M3) were quite different, which had significant effects on their photophysical properties, especially for two-photon excited fluorescence (2PEF). Under a high concentration (2×10−4 M), the compact aggregates of B3 and X3 exhibited enhanced 2PEF intensity versus fluorescence quenching of others. Moreover, B2, X2 and M2 presented higher polarity and lower cytotoxicity, while B2 and B3 with shorter PEG showed good performance in two-photon polymerization, indicating B2 will be a potential PS for 3D microfabrication of biosafety materials.

1. Introduction Photon technology has been widely used in many fields, such as data storage, fluorescence microscopy, MEMS fabrication and photodynamic therapy (PDT). However, due to the diffraction limit and low penetration depth, it is difficult to further improve the precision and spatial selectivity based on conventional linear absorption. The recently developed two-photon technology has great potential to solve the problem due to the advantages of higher spatial resolution and larger penetration depth of two-photon absorption (2PA) [1–7]. Moreover, because of the low damage of near infrared photons to biological tissue, the application of two-photon technology in biological field has attracted various attentions. At present, with the rapid developments of ultrafast laser setups and 2PA chromophores, two-photon fluorescence microscopy with ultra-high resolution has been widely used in bioluminescence imaging. Meanwhile, many complex three dimensional (3D) micro-structures, including 3D scaffolds for tissue engineering, have been fabricated by femtosecond laser direct writing based on twophoton photopolymerization (2PP) [8–13]. In vivo two-photon excited PDT (2PE-PDT) has also been conducted [14,15]. To obtain chromophores with enough 2PA capability and biocompatibility is an important prerequisite for the development of twophoton technology in biological field. Theoretically, 2PA is a third⁎

order nonlinear optical process. Hence, strategies which are helpful to improve the nonlinear optical effect have been introduced into the design of chromophores with a large 2PA coefficient, such as a long π conjugation system, strong electron donor or acceptor substituents [16–19]. In living organisms, chromophores need alternate contact with water in body fluid and lipid in various organelles, so a proper lipid-water amphiphilicity is essential. Without water solubility, chromophores cannot be transported to lesion sites with the body fluid. Meanwhile, without lipid solubility, chromophores cannot go through lipid membrane barriers and combine with certain organelles. Because most of chromophores with a long π conjugation system are generally lipid-soluble, to improve their water solubility has become an extensive research subject. The most common way is to chemically modify lipidsoluble chromophores with water-soluble groups [20–22]. Inevitably, lipid-water amphiphilic molecules usually exhibit surface activity and will form aggregates, which may affect the 2PA properties of chromophores and their application in biological fields. However, as we know, few relevant studies have been reported. Recently, a series of amphiphilic 2PA PSs by modifying benzylidene cycloalkanone moieties with different water-soluble groups were studied in our group, including carboxylate anionic, pyridyl cationic and polyethylene glycol (PEG) groups. Their potentials in 2PE-PDT [13,23–26] and 3D fabrication of hydrogels by 2PP [27,28] were

Corresponding author. E-mail address: [email protected] (Y. Zhao).

https://doi.org/10.1016/j.jphotochem.2018.12.017 Received 19 October 2018; Received in revised form 6 December 2018; Accepted 14 December 2018 Available online 14 December 2018 1010-6030/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. Chemical structures of six PSs.

water was measured by dynamic light scattering (DLS) using DynaPro NanoStar. 2PA cross-section (σ2) was determined using two-photon excited fluorescence (2PEF) method [30] and nonlinear transmission (NLT) method [31]. In 2PEF method, a Tsunami mode-locked Ti:sapphire laser system (720–880 nm, 80 MHz, < 130 fs) was used as the light source. the up-conversion fluorescence of all PSs in deionized (DI) water of 2 × 10−6 M and 2 × 10−4 M were measured using Rhodamine B in methanol of 2 × 10−8 M and 2 × 10−6 M as references, respectively. The up-converted fluorescence spectra were recorded by a monochromator (Omni-λ300) plus a photomultiplier (PMTH-S1CR131). In NLT method, the light source was a regeneratively amplified Ti:sapphire system (Spitfire F-1 K, 800 nm, 1 kHz, ∼130 fs). Samples were dissolved in DI with a concentration of > 2 × 10−3 M. Two large area (diameter 2 cm) photodiode detectors were used to record the powers of input beam and transmitted beam, respectively. More experimental details are provided in SI. In the prepolymer formulation for 2PP, six PSs were used as photoinitiators directly, while eosin was employed as the reference, monomer was the commercial water-soluble acrylate SR610, and DI water was used as solvent. The light source was a Tsunami Ti:sapphire laser (780 nm, 82 MHz, 80 fs). The sample was fixed on a xyz-step motorized stage and controlled by a computer (P-622.ZCL). An oilimmersion objective lens (100×, NA = 1.1, Olympus) was used to focus the laser beam into samples. After the fabrication, unpolymerized samples were washed off with DI water. Images of 2PP structures were observed by Nikon TI-U inverted microscope and recorded by Low temperature refrigeration high touch bar camera (PIXIS: 100B). In cytotoxicity experiment, HepG2 cells were seeded onto a 96 well plate at a density of 1 × 104 cells per well in 200 mL culture media and incubated for 24 h for cells to attach in an incubator in a humid atmosphere with 5% carbon dioxide at 37 °C, then the culture medium was replaced with PS-containing culture media. After that, the cells were incubated in the dark for 24 h, and washed with PBS before survival assessment by CCK-8 assay according to manufacturer’s protocol. The control group without PS was also treated in the same way and taken as the 100% cell survival base line.

confirmed. During the researches, some of these PSs were found to form aggregates in aqueous solutions. In this work, six PSs (shown in Fig. 1) with PEG modification were selected to investigate the effect of their aggregation behaviors on their photophysical properties. Some interesting results were obtained. Because PEG modification is widely used to improve the water solubility of organic compounds, we believe this work will be beneficial to follow-up studies. 2. Experimental 2.1. Materials Rhodamine B, eosin and other analytically pure reagents were purchased from Beijing Chemical Works. Chromatographically pure methanol was bought from Fisher Scientific Company and other chromatographically pure reagents were obtained from Xilong Chemical Co. Ltd. SR610 [polyethylene glycol (600) diacrylate] were from Sartomer Co. Ltd. Phosphate buffered saline (PBS) solution (pH = 7.4) and Dubach’s modified Eagle’s medium (DMEM, containing 4.5 g/L glucose, 100 unit/mL penicillin, 100 μg/mL streptomycin) were purchased from Beijing Solarbio Science & Technology Co. Ltd. Fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Co. Ltd. Cell Counting Kit-8 (CCK-8) and lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100) were from Beyotime Institute of Biotechnology. Six PSs were synthesized according to our previous reports [13,24] and purified by high-performance liquid chromatography (HPLC, LC6AD with a reverse phase C18 chromatography column of Shimapack VP-ODS) to ensure the purity of > 99%, as shown in Fig. S1 in Supporting Information (SI). Among them, B2, B3, X2 and X3 are cyclopentanone derivatives, M2 and M3 are cyclobutanone derivatives. All of PSs are modified with two PEG chains, triethylene glycol for B2 and B3, and tetraethylene glycol for the other four. Additionally, B2, X2 and M2 are symmetrically modified with two PEG chains on both sides of the π conjugated structure, respectively, while B3, X3 and M3 are asymmetrically modified with two PEG chains on one side of the π conjugated structure. 2.2. Methods

3. Results and discussions UV-vis spectra of PSs were obtained on a Hitachi U-3900 spectrophotometer, and their steady-state fluorescence spectra were recorded on Hitachi F-4500 fluorescence spectrophotometer. Fluorescence quantum yield (Φ) was measured in diluted solutions according to the literature, Rhodamine B in methanol was used as reference (Φr = 0.7) [29]. Surface tension of PSs in DI water solutions with different concentrations were measured by Wilhelmy plate method using a dynamic contact angle tensiometer (DCAT21). Particle size of PSs’ aggregation in

3.1. Water-solubility and polarity Six PSs can dissolve well in DI water and the solubility of B2 and B3 are 2.72 mM and 5.36 mM, respectively, while the corresponding data of other four PSs are all more than 20 mM due to their longer PEG chains. However, after the aqueous solutions were filtered with 0.2 μm microporous filters, most of PSs couldn’t pass through the filter, which 207

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PSs within 2 × 10−6−2 × 10−4 M, which indicates that it has the lowest degree of aggregation.

Table 1 Solubility in DI water, the retention times in HPLC (mobile phase: 85% methanol in DI water) and linear photophysical properties in different solvents of six PSs. PSs

B2

B3

X2

X3

M2

M3

Solubility (mM) Retention time (min)

2.72 8.3 507

5.36 13.1 511

28.9 8.0 508

26.7 12.4 513

> 100 6.7 518

> 100 9.6 527

4.38 640

4.13 638

4.39 641

3.71 642

3.98 659

3.75 657

5.85 461

4.39 462

6.66 468

4.49 466

7.48 504

4.35 498

abs λmax in water (2 × 10−6 M) (nm) εmax in water (104 M−1 cm−1) fl λmax in water (2 × 10−6 M) (nm) Φf in water (10−3)

abs λmax in water (2 × 10−4 M) (nm) abs λmax in DMF (2 × 10−6 M) (nm) abs λmax in

n-octanol (2 × 10 (nm)

−6

M)

472

475

472

475

488

493

482

485

482

487

489

494

3.3. Surface tension The relationship between the surface tensions of the PS solution and their concentration in DI water was investigated. As showed in Figs. 3 and S5, all PSs have surface activity and form aggregates within the measure range. The aggregation behaviors of symmetrical PSs (B2, X2 and M2) and asymmetrical PSs (B3, X3 and M3) are quite different. The surface tension of the solution containing symmetrical PSs decline more slowly in lower concentration range but faster in higher concentration range, while the corresponding trend is opposite for asymmetrical PSs. Only one critical micelle concentration (CMC) can be obtained for B3 (3.6 × 10−6 M) and X3 (5.3 × 10−6 M), but the plot of surface tension verses concentration seems more complicated for the other PSs, which indicates that their aggregation forms are changing constantly.

abs λmax is the maximum absorption wavelength in solutions; εmax is the molar abs fl absorption coefficient at λmax ; λmax is the maximum fluorescence emission wavelength at the excitation of 510 nm; and Φf is the fluorescence quantum yield.

3.4. Dynamic light scattering Based on the above results, it is clear that aggregates are formed for all PSs when the concentration exceeds 2 × 10−4 M. To characterize the aggregates, dynamic light scattering (DLS) of six PSs with two concentrations were conducted. Besides 2 × 10−4 M for all PSs, 2.5 × 10−2 M was used for X2, X3, M2 and M3, while 2.5 × 10−3 M and 5.0 × 10−3 M were used for B2 and B3, respectively, due to their limited water solubility. As listed in Table 2, all PSs form small aggregates with average sizes of 6−11 nm at 2 × 10−4 M, except M2, whose average size is 42 nm. At high concentrations of 10-3−10−2 M, two kinds of aggregates (small ones with average sizes of 3–5 nm and big ones with average sizes of 87−441 nm) can be formed simultaneously, which indicates that there are multiple forms of aggregation in the solutions. Interestingly, M2 is still an exception that only one kind of aggregates with an average size of 33 nm can be found. For it has the highest polarity, the concentration of 2.5 × 10−2 M may be still not enough for M2 to form larger aggregates.

implied that these PSs were not unimolecular dispersion but formed some kinds of aggregates in the solutions. As listed in Table 1, the retention times of symmetrically modified PSs (B2, X2 and M2) are shorter than those of asymmetrical PSs (B3, X3 and M3). For a reverse phase C18 column was used in this study, it indicated that B2, X2 and M2 have higher polarity than B3, X3 and M3, respectively. Moreover, the polarity of these PSs enhance with the length increase of the PEG chain. M2 and M3 with cyclobutanone as the center moiety are more soluble than the other four PSs, which is consistent with their polarity. 3.2. Linear photophysical property The normalized UV–vis absorption spectra and the florescence emission spectra of six PSs in DI water (2 × 10−6 M) are shown in Fig. S2, their corresponding linear photophysical data are listed in Table 1, which indicates that the different length and modification position of abs PEG chains have no significant effects on absorption peaks (λmax ) and fl emission peaks (λmax ) of these PSs. However, the absorption spectra of M2 and M3 with cyclobutanone as the center moiety are obviously different from the others’, exhibiting a shoulder absorption peak around fl 450 nm and a clearly red shift of λmax . Additionally, the symmetrical PSs (B2, X2 and M2) exhibit slightly higher molar absorption coefficient (εmax ) and fluorescence quantum yield (Φf) than those of asymmetrical ones (B3, X3 and M3). When the concentrations of these PSs in DI water increased gradually, their linear absorption spectra all presented a significant blue shift, as shown in Figs. 2 and S3. An interesting phenomenon can be seen that the blue-shift trend of symmetrical PSs (B2, X2 and M2) are quite different from those of asymmetrical PSs (B3, X3 and M3). In the plot of the maximum absorption peak versus concentration (Figs. 2 and S4), it is very clear that the absorption peaks of all PSs are stable with a certain range of low concentrations. However, once the concentration exceeds a limit (∼10−5 M), an obvious mutation of the absorption peak appears for asymmetrical PSs (B3, X3 and M3) and a new stable platform will reach soon (over 10−4 M), while a gradual blue-shift keeps going on during the measure range for symmetrical PSs (B2, X2 and M2). When compared with the absorption peaks of PSs in different solvents abs (Table 1), the λmax values of these PSs in water at high concentration are similar to those in dilute organic solvents, such as DMF and n-octanol. A proposed reason is that these PSs would form aggregates in water when the concentration is high enough, so that a local microenvironment similar to organic solvent is built around themselves. Additionally, asymmetrical PSs (B3, X3 and M3) are easier to form compact aggregates compared with symmetrical ones (B2, X2 and M2). With the highest polarity, M2 exhibits the shortest blue-shift among all

3.5. Up-conversion florescence The up-conversion fluorescence spectra of these PSs excited by 800 nm femtosecond laser at two concentrations (2 × 10−4 M and 2 × 10−6 M) were investigated. As shown in Fig. 4, at low concentration (2 × 10−6 M), the up-conversion fluorescence intensities of B2, X2 and M2 are much higher than those of B3, X3 and M3, respectively. However, at high concentration (2 × 10−4 M), the relation is completely reversed for B2 versus B3 and X2 versus X3, while the up-conversion fluorescence intensities of M2 and M3 are both decreased. As depicted in Fig. S2, the overlaps between the linear absorption and fluorescence emission spectra of six PSs at the concentration of 2 × 10−6 M are very small. Additionally, as discussed in Section 3.2, when the concentrations of these PSs in DI water reached ∼2 × 10−4 M, their linear absorption spectra all presented a significant blue shift, so the inner-filtering effects for these PSs was very limited within the measured range of 2 × 10-4−2 × 10−6 M. The reason for the decreased emission of B2, X2, M2 and M3 at the high concentration should be due to aggregation induced fluorescence quenching. However, B3 and X3 present enhanced fluorescence intensity when compared with the upconversion fluorescence intensity of Rhodamine B in methanol. We speculate it may be due to aggregation induced fluorescence enhancement when the compactness of the aggregates is high enough. 3.6. 2 PA cross-section Using Rhodamine B in methanol as reference, the σ2 values of these PSs at 800 nm were measured by both 2PEF method and NLT method. As listed in Table 2, the σ2 values measured by two methods are 208

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Fig. 2. Normalized UV–vis absorption spectra (upper) and the plot of the maximum absorption peaks versus concentrations (lower) of B2 and B3 in DI water.

Fig. 3. The plot of surface tensions versus concentrations of the DI solutions containing B2 and B3.

obviously different. The data from 2PEF are lower than those from NLT. Because the up-conversion fluorescence intensity of these PSs can fluctuate significantly due to the formation of aggregates, the measurement errors are relatively larger by 2PEF method. On the contrary, the data of six PSs obtained by NLT method are comparable to the data of a homologous PS (Y4, 287 GM at 800 nm) [27], which is modified by four sodium carboxylate groups and confirmed having no aggregates in DI water (Fig. S6).

Table 2 Average sizes of aggregates and 2 PA cross-section (σ) of PSs in DI water at 800 nm. PSs

B2

B3

X2

X3

M2

M3

Average size of aggregates at 2 × 10−4 M (nm) Average sizes of two kinds of aggregates, small ones/big ones, at 10−3−10−2 M (nm) σ measured by 2PEF method at 2 × 10−6 M (GM) σ measured by 2PEF method at 2 × 10−4 M (GM) σ measured by NLT method at > 2 × 10−3 M (GM)

8

6

11

8

42

9

5/ 166

3/ 441

5/ 166

3/ 87

33

3/ 87

148

46

174

83

121

72

17

199

17

182

0.2

0.3

263

256

381

266

247

140

3.7. Two-photon polymerization All of these PSs can be used as a photoinitiator directly for 2PP in water solution. For the formula of 79.87 wt% SR610 + 19.97 wt% DI water + 0.16 wt% PS, low 2PP threshold energies for six PSs were obtained as shown in Fig. 5, wherein Eth is defined as the lowest laser power at focus point that can fabricate a solid line. The corresponding Eth data for six PSs are much lower than that of the commercial PS eosin 209

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Fig. 4. Up-conversion fluorescence spectra of six PSs at 2 × 10−6 M (left) and 2 × 10−4 M (right) under the excitation of 800 nm femtosecond laser.

4. Conclusion

(2.18 mW) and the differences between them are quite small. However, the solid lines obtained by the formula containing PSs with longer PEG chains tended to collapse due to their strong water absorption capability, which decreased the mechanical strength of the polymerized structures.

The effect of the aggregation behavior of lipid-water amphiphilic PSs on their linear and nonlinear photophysical properties were studied based on a series of PEG modified benzylidene cycloalkanone PSs. The results showed that various aggregates could form for these PSs with the increase of concentration, but the aggregation behavior of symmetrically modified PSs (B2, X2 and M2) and asymmetrically modification PSs (B3, X3 and M3) were quite different. Asymmetrical PSs were easier to form compact aggregates compared with symmetrical PSs. Under high concentration, the compact aggregates of B3 and X3 exhibited enhanced 2PEF intensity versus fluorescence quenching of the others. Additionally, symmetrical PSs (B2, X2 and M2) had higher polarity and lower cytotoxicity. B2 and B3 with shorter PEG showed good performance in 2PP. B2 presented a great potential for 3D microfabrication of biosafety materials.

3.8. Cytotoxicity Cytotoxicity is an important factor for PSs used in biological fields, such as preparation of 3D hydrogel scaffold for tissue engineering. The cytotoxicity of the six PSs (20 μM) toward HepG2 cells were investigated by CCK-8 assay. As shown in Fig. 6, B3, X3 and M3 exhibit obvious cytotoxicity, while the other three PSs (B2, X2 and M2) are relatively safe, exhibiting more than 90% cell viability at this concentration. Combining with our previous report, the cytotoxicity of a PS is positively related to its cellular uptake [13]. The higher the polarity of a Ps, the harder it is to pass through the cell membrane, so that a good biological safety can be achieved for higher polarity PSs of B2, X2 and M2.

Fig. 5. Solid lines fabricated using formulations containing PSs by 800 nm femtosecond laser direct writing based on 2PP. 210

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Fig. 6. Cytotoxicity of PSs toward HepG2 cells. The error bars denote standard deviation from three replicates.

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