Uptake of Chlorin e6 Photosensitizer by Polystyrene-Diphenyloxazole-Poly(N-Isopropylacrylamide) Hybrid Nanosystem Studied by Electronic Excitation Energy Transfer

Polystyrene (PS)-diphenyloxazole (PPO) nanoparticles with attached cross-linked poly-N-isopropylacrylamide (PNIPAM) chains were obtained resulting in PS-PPO-PNIPAM hybrid nanosystems (NS). Fluorescence spectra of chlorin e6 added to PS-PPO-PNIPAM hybrid NS revealed electronic excitation energy transfer (EEET) from PS matrix and encapsulated PPO to chlorin e6. EEET efficiency increased strongly during 1 h after chlorin e6 addition, indicating that uptake of chlorin e6 by PNIPAM part of hybrid NS still proceeds during this time. Heating of PS-PPO-PNIPAM-chlorin e6 NS from 21 to 39 °C results in an enhancement of EEET efficiency; this is consistent with PNIPAM conformation transition that reduces the distance between PS-PPO donors and chlorin e6 acceptors. Meanwhile, a relatively small part of chlorin e6 present in the solution is bound by PNIPAM; thus, further studies in this direction are necessary.


Background
The main disadvantage of the photodynamic therapy of cancer is the low depth of the excitation beam penetration into the tissue [1]. Thus, radiodynamic therapy approach to cancer treatment (where sensitizer could be efficiently excited with the X-rays able to penetrate deep into the body) was proposed [2] and is intensively studied in the last years [3][4][5]; one of the research scopes is the development of sensitizers that generate singlet oxygen upon X-ray excitation [5][6][7][8]. The key process in such X-ray sensitizer is the electronic excitation energy transfer (EEET) between its scintillating and sensitizing components [2,6,[9][10][11]. Another important component of the mentioned X-ray sensitizer is the way of keeping scintillating and sensitizing parts together at the distance optimal for EEET; chemical conjugation [2,3], electrostatic attraction [8,12], surfactant [11], or polymer shell [4] could be mentioned. Earlier, in the frames of designing nanosystems (NS) for X-ray excited sensitizing of singlet oxygen, we studied EEET in polystyrene (PS)-diphenyloxazole (PPO)-chlorin e 6 NS, where photosensitizer chlorin e 6 was bound to PS-PPO nanoparticle (which can be used as scintillator [13,14]) via surfactant (sodium dodecylsulphate) shell [15].
Poly(N-isopropylacrylamide) (PNIPAM) belongs to stimuli-responsive materials that change their properties in response to internal or external stimulus [16]. Linear PNIPAM is known to undergo conformational transition upon heating, i.e., the polymer shrinks (due to becoming hydrophobic and thus expelling water molecules) at the temperatures over the lower critical solution temperature (LCST) that equals 32°C for linear PNIPAM [17]. Considerable decrease in the PNIPAM shell width upon temperature transition over LCST was shown for PS-PNIPAM nanoparticles in [17]. At the same time, for the PNIPAM chains conjugated to dextran, the temperature of conformation transition was shown to be 2-4°C higher as compared to linear PNI-PAM of similar molecular weight and polydispersity due to the steric interaction between PNIPAM chains hindering the conformation transition [18].
In this work, PS-PPO nanoparticles covered by cross-linked PNIPAM shell (Figs. 1 and 2) were obtained resulting in PS-PPO-PNIPAM hybrid NS. The possibility of using cross-linked PNIPAM for attaching chlorin e 6 sensitizer to PS-PPO NP scintillator was studied. This polymer has the conformational transition at physiological temperatures (for cross-linked PNIPAM, LCST should be higher than 32°C). Thus, shrinking of cross-linked PNIPAM network following heating under excitation could result in decrease of the distance between PS-PPO donor and chlorin e 6 acceptor that would increase the efficiency of EEET and thus the efficiency of tumor destruction.

Synthesis and Characterization of Nanosystems
Polystyrene-poly(N-isopropylacrylamide) hybrid nanosystems, doped with PPO (PS-PPO-PNIPAM hybrid NS), were synthesized as follows. First, PS-co-PNIPAM core nanoparticles doped with PPO were prepared by microemulsion polymerization [13,14,19,20]. Briefly, 0.2 g of NIPAM, 0.2 g of sodium dodecylsulphate, and 0.01 g of NaH 2 PO 4 × H 2 O were dissolved in 90 g of H 2 O. Then, 0.09 g of PPO was dissolved in 1.8 g of styrene and the obtained mixture was added dropwise during 30 min, too. The mixture was stirred at 700 rpm, and Ar was bubbled into the mixture for 30 min. After the temperature increased to 70°C, 0.01 g of K 2 S 2 O 8 dissolved in 1 ml of H 2 O was injected to initiate the polymerization. Secondly, PNIPAM shell layer was fabricated after 4 h heating at 70°C. For this purpose, aqueous solution of monomer NIPAM (0.69 g) and cross-linker N,N′-methylenebisacrylamide (BIS) (0.06 g) were added into the reactor using a syringe. The reaction was allowed to continue for 3 h at 70°C and additional 1 h at 90°C. The mixture was cooled to room temperature and dialyzed during 48 h using cellulose membrane with MWCO 3500 Da.
Transition electron microscopy (TEM) images of the obtained nanosystems are presented in Fig. 2. For the sample preparation, 400 mesh Cu grids with plain carbon film were rendered hydrophilic by a glow discharge treatment (Elmo, Cordouan Technologies Bordeaux France). A 5-μl drop was deposited and let adsorbed for 1 min, then the excess of solution was removed with a piece of filter paper. The observations of the PS-PPO-PNIPAM nanosystems were carried on two TEMs, Tecnai G2 or CM12 (FEI, Eindhoven, Netherlands), and the images were acquired with a ssCCD Eagle camera on the Tecnai and a Megaview SIS Camera on the CM12. It is seen from Fig. 2 that the obtained hybrid nanosystems consist of several bound spherical PS-PPO NP; we believe that they are bound by cross-linked PNIPAM polymer net. Thus, PS-PPO-PNIPAM hybrid nanosystems were obtained.

Spectral Measurements and Sample Preparation
Absorption spectra were measured using a Specord M40 spectrophotometer (Carl Zeiss, Germany). Fluorescence excitation and emission spectra were registered with the help of a Cary Eclipse fluorescent spectrophotometer (Varian, Australia). Absorption and fluorescence measurements were performed in 1 × 1 cm quartz cell at room temperature. Fifty millimolars of TRIS-HCl buffer, pH 7.2, was used as a solvent. For spectral measurements, the obtained solution of PS-PPO-PNIPAM hybrid NS was dissolved 100 times in buffer. Stock solution of chlorin e 6 at the concentration 10 mM was prepared in DMF and further diluted in buffer to 1 mM concentration. Small aliquot of this 1 mM solution of chlorin e 6 was then added to 100 times dissolved buffer solution of NS; the final concentration of chlorin e 6 was 2 μM, and DMF admixture was thus 0.02%. Fluorescence excitation and emission spectra of PS-PPO-PNIPAM hybrid NS solution with chlorin e 6 was measured in 0, 5, 10, 20, 40, 60, 80, and 100 min after the addition of chlorin e 6 to the solution of PS-PPO-PNIPAM hybrid NS. In nearly 80 min, the saturation was reached.
For temperature-dependent measurements, solution of PS-PPO-PNIPAM hybrid NS in the presence of chlorin e 6 was placed into the thermostatted cell holder (T = 23°C). In 88 min after the preparation of the sample, after the uptake of chlorin e 6 by PS-PPO-PNIPAM hybrid NS reached saturation, water flow from the water bath was switched on heating the sample to 39°C (this temperature should exceed LCST for the cross-linked PNIPAM). Fluorescence excitation spectra of chlorin e 6 (emission at 680 nm) were then measured in different time intervals after the heating was started. Experimental results state that the conformation transition starts in about 3 min after the heating was started. It should be mentioned that in about 18 min after the heating was started, coagulation of the NS occurred giving macroscopic clots (which however disappear after the solution was cooled down, thus coagulation is reversible). Temperature experiment was performed three times, and similar tendencies were obtained.

Results and Discussion
Absorption, fluorescence excitation, and fluorescence emission spectra of the obtained PS-PPO-PNIPAM hybrid nanosystems in 50 mM Tris-HCl buffer (pH 7.2) are presented in Fig. 3. Absorption spectrum contains the bands corresponding to styrene (maximum near 260 nm) and PPO (maximum near 307 nm). At the same time, fluorescence emission spectrum upon excitation at 250 nm (the range of styrene absorption) resulted in the spectrum that belongs exclusively to PPO (with maximum at 367 nm), while the emission of styrene (due at 307 nm [15]) was not observed. Thus, EEET from styrene to incorporated PPO is near to complete. EEET is also supported by the excitation spectrum of PPO emission (380 nm) where styrene band near 260 nm is clearly observed (Fig. 3). It should be added that clear vibronic structure could be observed in PPO emission spectrum (which is not observed for the PPO  water solution [14]) that additionally points to the PPO incorporation into the PS matrix.
Further, to the solution of the obtained PS-PPO-PNIPAM hybrid nanosystems in 50 mM Tris-HCl buffer (pH 7.2), the photosensitizer chlorin e 6 was added. Absorption spectrum of chlorin e 6 shows almost no change in the presence of hybrid nanosystems as compared to buffer solution, except small decrease of the optical density in the maxima (Fig. 4).
At the same time, effect of the addition of chlorin e 6 to PS-PPO-PNIPAM hybrid NS on fluorescence spectra is much more noticeable as compared to absorption ones. First of all, the presence of chlorin e 6 leads to the quenching of PPO fluorescent emission of PS-PPO-PNIPAM hybrid NS, and this quenching does enhance during about an hour (Fig. 5). Generally, such quenching could be due to either EEET (that would lead to the quenching of donor emission at all emission wavelengths) or reabsorption (that would lead to the quenching of donor emission at the wavelengths of the acceptor absorption). Differences of PPO emission spectra (Fig. 5) show that immediately after chlorin e 6 addition, the contribution of reabsorption to PPO emission quenching is significant (but EEET also takes place). At the same time, the contribution of reabsorption further decreases with time and this of EEET grows.
Another effect of addition of chlorin e 6 to PS-PPO-PNIPAM hybrid NS is the appearing of chlorin e 6 emission (upon excitation of PS at 250 nm) that is shifted to the long-wavelength region as compared to the one of free chlorin e 6 ; both intensity and shift do increase with time and reach saturation in about an hour (Fig. 6). This shift points to the influence of the PNI-PAM surrounding on the bound molecules of chlorin e 6 .
At the same time, emission of chlorin e 6 upon excitation to its own absorption (at 400 nm) is only slightly changed in the presence of PS-PPO-PNIPAM NS. Together with the small change in chlorin e 6 absorption (Fig. 4), this means that only small part of chlorin e 6 molecules is bound to PS-PPO-PNIPAM NS.
Finally, the PS-PPO band appears in excitation spectrum of chlorin e 6 added to PS-PPO-PNIPAM hybrid NS (emission at 680 nm, where the contribution of   the PNIPAM-bound chlorin e 6 to the total emission of chlorin e 6 is close to maximum). This band is weak at first, but further, its intensity strongly increases with time ( Fig. 7). At the same time, this PS-PPO band is very weak in the excitation spectrum of chlorin e 6 upon emission at 660 nm (i.e., at maximum of the free chlorin e 6 spectrum) even after 100 min after the addition of chlorin e 6 .
Thus, chlorin e 6 binds to PS-PPO-PNIPAM hybrid NS that causes EEET from PS matrix and encapsulated PPO to chlorin e 6 . EEET efficiency enhances with time (during about an hour after chlorin e 6 addition), indicating that uptake of chlorin e 6 by PNIPAM network of PS-PPO-PNIPAM hybrid NS still proceeds during this time. At the same time, a relatively small part of chlorin e 6 present in the solution is bound by PNIPAM.
It should be also mentioned that PS-PPO NP were shown to emit the fluorescence of PPO [14] or attached porphyrin [13] when excited with X-rays. Thus, EEET from PS to chlorin e 6 observed in PS-PPO-PNIPAM hybrid NS under the UV excitation of PS means that the energy of X-rays could be also transferred to chlorin e 6 in the studied NS. At the same time, rather high concentration of PS-PPO NP is required for the direct observation of X-ray stimulated emission of such NP [13,14]. One of the ways to increase the sensitivity of such NS to X-rays could be the addition of components containing heavy atoms.
PNIPAM is known to experience the conformation transition at LCST equal to 32°C. For the cross-linked polymer, the LCST value should be still higher. If our PS-PPO-PNIPAM hybrid NS consists of cross-linked PNIPAM network surrounding PS-PPO NPs, and chlorin e 6 is bound to PNIPAM network, we could expect the decrease of PPO-chlorin e 6 distance and thus the increase of EEET efficiency upon heating the whole hybrid NS.
To verify this idea, the effect of heating of PS-PPO-PNIPAM hybrid NS in the presence of    9 The dependence on the time after the heating to 39°C was switched on of the ratio of the emission intensity of chlorin e 6 added to PS-PPO-PNIPAM hybrid NS at 680 nm upon excitation at 320 nm (absorption by PPO with further EEET to chlorin e 6 ) to that upon excitation at 404 nm (direct excitation of chlorin e 6 ). Error bars account for the noise of the registered excitation spectra chlorin e 6 on fluorescence excitation spectra of chlorin e 6 was studied. Heating experiment was performed three times; similar tendencies were demonstrated. Results of one of these experiments are presented in Figs. 8 and 9. Thus, the solution of PS-PPO-PNIPAM hybrid NS in the presence of chlorin e 6 was heated to the temperature of 39°C (this temperature should exceed LCST for the cross-linked PNIPAM); the heating started after the uptake of chlorin e 6 by PS-PPO-PNIPAM hybrid NS reached saturation. During the heating, the ratio of chlorin e 6 fluorescence intensities (emission at 680 nm) upon excitation at 320 nm (I ex320 ; that is chlorin e 6 emission due to PPO-to-chlorin e 6 EEET) and 404 nm (I ex404 ; that is chlorin e 6 emission upon excitation to its own Soret band) was studied (Figs. 8 and 9); we believe this ratio to reflect the efficiency of PPO-to-chlorin e 6 EEET. Thus, the first 3 min after heating started, I ex320 /I ex404 decreased from 0.9 to 0.85, perhaps due to the decrease of the chlorin e 6 -to-PNIPAM binding affinity upon temperature increase. Further, the value of I ex320 /I ex404 ratio increased up to 1.02, which was accompanied by the increased light scattering (Fig. 8; scattering is manifested as the intensity increase near 660 nm). This could be explained by conformation transition in PNIPAM that leads to reducing of cross-linked PNI-PAM network volume. This causes the decrease of the distance between PS-PPO donor and chlorin e 6 acceptor molecules bound to PNIPAM network and thus to increasing of EEET efficiency.
In summary, processes in the solution of PS-PPO-PNIPAM hybrid NS in the presence of chlorin e 6 addition could be described as follows. First, addition of chlorin e 6 to the solution of PS-PPO-PNIPAM hybrid NS in buffer (Fig. 10, process 1) leads to the penetration of the small part of chlorin e 6 into the PNIPAM network of the hybrid NS; this resulted in EEET from PS and PPO to chlorin e 6 . Such penetration proceeds during about an hour accompanied with the increase in EEET efficiency (Fig. 10, process 2). Further heating of the sample to the temperature exceeding LCST results in the conformation transition of the PNIPAM network that leads to the decrease of the distance between PS-PPO and chlorin e 6 molecules, and thus to still higher increase of PS-PPO-to-chlorin e 6 EEET efficiency (Fig. 10, process 3).

Conclusions
The fluorescent study revealed the uptake of chlorin e 6 by PS-PPO-PNIPAM hybrid NS as well as electronic excitation energy transfer from the PS matrix via encapsulated PPO to chlorin e 6 ; uptake reached saturation in about an hour.
Heating of PS-PPO-PNIPAM-chlorin e 6 NS from 21 to 39°C results in enhancement of EEET efficiency; this is consistent with PNIPAM conformation transition that reduces the distance between PS-PPO donors and chlorin e 6 acceptors.
Meanwhile, a relatively small part of chlorin e 6 present in the solution is bound to PNIPAM; thus, further studies in this direction are necessary. Funding This work was supported by the Ministry of Education and Science of Ukraine (program "Science in Universities", project no. 16БФ051-03 "The study of electronic-vibrational processes in composites and nanosystems promising for solar energetics, low-power-consuming light sources and medicine").

Availability of Data and Materials
The data supporting the conclusions of this article are included within the article.
Authors' Contributions ML carried out spectral measurements and wrote the article. LV and ON performed the synthesis of nanosystems. NK performed TEM characterization of nanosystems and participated in the discussion. VY participated in the design of the study and discussion of the results and coordination. All authors read and approved the final manuscript.

Competing Interests
The authors declare that they have no competing interests.

Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.