Two-photon absorption of polyfluorene aggregates stabilized by insulin amyloid fibrils

University of California, Center for Oligo Sciences Building North, Santa Barbara, US Chalmers University of Technology, De Engineering, Kemivägen 10, Gothenburg, 41 Wroclaw University of Technology, Advanc Group, Faculty of Chemistry, 50-370, Wrocl University of South Australia, Ian Wark R Australia 5095, Australia † Electronic supplementary informa 10.1039/c5ra08302h Cite this: RSC Adv., 2015, 5, 49363


Introduction
Conjugated polymers have attracted much attention as effective probes for amyloid protein bril recognition and are being developed for imaging, diagnostic and therapeutic applications 1 . Their tunable photophysical properties in the visible and near-infrared spectrum, which is particularly sensitive to protein conformation upon binding, allow for monitoring of the brillization with a variety of optical techniques 2 . Among those, techniques based on nonlinear optics are especially promising as they can improve penetration depth, reduce photo-bleaching and minimize photo-damage of biological systems. However, in order to use multiphoton based techniques molecules with large two-photon absorption cross-sections are required that enable efficient excitation in the near infrared region, which is safe for biomolecules. Polyuorenes (PFO) have the potential to be interesting markers since they exhibit sizeable multiphoton absorption properties, 3 which may facilitate amyloid bril detection and imaging by less invasive multiphoton optical methods. 4 In the context of binding to biomolecules, two groups of compounds are of particular interest: water-soluble conjugated polyelectrolytes and non-water-soluble conjugated polymers or oligomers. In case of the rst group the functional charged groups enhance the interaction with amyloids due to electrostatic binding with charged residues in the protolaments of the brils 5 , whereas for the second group only weaker (stacking type) interaction occurs that is sensitive to small structural changes and the mode of interactions with the brils.
In this letter we explore complexation and the mechanism of molecular interactions of a non-water-soluble polyuorene derivative composed of a backbone with ethylene glycol side chains (Fig. 1a) by investigating its photophysical properties and interaction with amyloid brils, most likely mediated by weak forces such as van der Waals and p-stacking 6 . Two forms of polyuorene derivative were studied: (i) PFO dissolved in ethanol and (ii) PFO that has aggregated upon addition of waterbased buffer. Then we studied the optical signatures of both forms of the polyuorene derivative with insulin protein that is known to self-assemble into amyloid brils under denaturing conditions such as low pH and high temperature. Insulin brils predominantly consist of b-sheet structures and resemble the structures of diseases related proteins such as tau, Ab (1-42) (Alzheimer's) and a-synuclein (Parkinson's) 7 or prions (Creutzfeldt-Jakob's). 8 The key nding of our work is that PFO aggregation can be stabilized in solution upon binding to insulin amyloid brils and that can lead to a signicant enhancement of two-photon absorption.

Materials
Polyuorene derivative with polyethylene glycol side chains (PFO) was synthesized as follows: 2,7-dibromo-9,9-bis(2-(2-methoxy ethoxy)ethyl)-9H-uorene (0.264 g, 0.5 mmol), 2,2 0 -(9,9-bis(2-(2methoxyethoxy)ethyl)-9H-uorene-2,7-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane) (0.311 g, 0.5 mmol), K 3 PO 4 tribasic (1.06 g, 5 mmol), aliquot 336 (3 drops) and water (2.3 mL) were dissolved in toluene (15 mL) and nitrogen gas was bubbled for 20 minutes. Pd 2 (dba) 3 (9 mg, 0.01 mmol) and P(o-tolyl) 3 (12 mg, 0.04 mmol) were added to the reaction mixture and nitrogen gas bubbling continued for an additional 25 minutes. The reaction mixture was heated at 90 C for 1 hour and 20 minutes. The oligomer solution was precipitated by drop-wise addition of hexane and the solid was collected by ltration. The oligomer was re-dissolved in THF and passed through short silica gel column using THF as eluent. The oligomer solution was concentrated on a rotavapor and precipitated by drop-wise addition of hexane and the solid was collected by ltration. The oligomer was further puried by Soxhlet extraction with hexane and then washed out with THF. The volume of the THF extract was reduced by evaporation and precipitated with hexane. The solid collected was dried at 40 C in a vacuum oven for an overnight to give a yellowish powder (0.3 g, 81%). The synthesis scheme is presented in Fig. S1 † together with NMR spectra (Fig. S2 and S3 †). Characterization by gel permeation chromatography (GPC) indicates that PFO has low numberaverage molecular weight of Mn $3.3 kg mol À1 and a high polydispersity index PDI $6.2. In average there is 9 repeating units within the oligomer chain.
Amyloid brils were prepared as follows: monomer insulin protein from bovine pancreas was purchased from Sigma Aldrich, dissolved in pH ¼ 2 (0.01 M HCl) water buffer and adjusted to nal concentration of 5 mg mL À1 and used without further purication. Prepared solutions were then heated at 65 C for 24 hours. Fibrils formation was tested by Thioavine T (ThT) standard with monomer proteins as references (Fig. S4 †).
Amyloid brils-oligomer complexes were prepared as follows: lyophilized polyuorene was partially dissolved in pure ethanol and concentration was calculated to be approximately 2 g L À1 (molar extinction coefficient in ethanol 3 ¼ 4500). In a next step polyuorene was either added directly from ethanol to solution with insulin brils and further studied with absorption techniques or it was mixed in volume ratio 1 : 10 with water buffers rst in order to initiate formation of aggregates.

Methods
Size exclusion chromatography (SEC) was performed on Waters 510 HPLC pump with a Waters 486 UV detector (254 nm). The system runs at room temperature with THF as eluent, 0.5 mL min À1 . The column is a Agilent PLgel 5 mm MIXED-C. The concentration of the sample was 0.5 mg mL À1 , which was ltered (lter: 0.45 mm) prior to the analysis. The relative molecular masses were calculated by calibration relative to polystyrene standards. 1 H NMR and 13 C NMR spectra were acquired from a Varian Inova 400 MHz NMR spectrometer. Tetramethylsilane was used as an internal reference with deuterated chloroform as solvent. UV-Vis spectroscopy. Absorption spectra were recorded on a Jasco V-670 spectrophotometer, using solvent as a baseline. Linear dichroism spectra were recorded on a Chirascan CD spectrophotometer equipped with an LD accessory unit.
Fluorescence was studied using a Hitachi F-4500 uorescence spectrophotometer. The samples were excited at l ex ¼ 375 nm where the maximum of absorption of PFO was in ethanol and emission was recorded in spectral range from 390 nm to 650 nm.
Z-scans for two-photon absorption (TPA). The Z-scan technique is used to measure nonlinear refractive index (n 2 ) and nonlinear absorption coefficient (b). Both quantities can be measured simultaneously by monitoring changes in transmittance of a focused laser beam as the sample travels in the zdirection. In order to measure TPA that is attributed only to PFO-bound to amyloid brils rst the detection limit for dissolved PFO was experimentally determined by gradual lowering of oligomer concentration by diluting it in ethanol. No open aperture (OA) signal was detectable around 0.5 mg mL À1 and such concentration of PFO was used for further experiments on TPA with amyloid brils. Samples for measuring nonlinear absorption were mixed in volume ratio 1 : 4 (insulin amyloid : polyuorene) where insulin brils stock solution was used (5 mg mL À1 ) and polyuorene at concentration 0.5 mg mL À1 . Obtained results on the cells with PFO, insulin brils with PFO and insulin brils with aggregates of PFO were calibrated against closed aperture Z-scan measurements performed on a fused silica plate and compared with the measurements on an identical glass cell lled with the solvents alone: mixture of ethanol/pH ¼ 2 water buffer for simultaneous recording of standard open-aperture (OA). The traces of OA scans obtained by dividing each of them by the laser input reference were analyzed with the help of a custom tting program.

Results and discussion
In a rst set of experiments the photophysical properties of PFO were investigated by absorption and uorescence spectroscopy. PFO displayed good solubility in ethanol, which was estimated to be at least 2 g L À1 at room temperature. In contrast, PFO was found to be non-soluble in water-based buffers. The good miscibility of ethanol and water permitted us to record the absorption and emission spectra of dissolved and aggregated PFO through solvent exchange. Aggregation resulted in absorption redshiing and considerable broadening of the spectrum. These results indicate that the polyuorene structure and conformation are inuenced by acidied water based conditions. Similar photophysical features have been attributed to formation of aggregates and reported previously for poly-uorenes 9 . We note that a gradual redshi in peak absorption by up to 50 nm is accompanied by a decrease in absorption strength and the appearance of a long tail above 500 nm which can be explained with light scattering by the aggregates (Fig 1b). We observe also a shoulder at 350-400 nm which arises due to the fraction of PFO that remains dissolved. The redshi of the absorption maximum occurs due to conformational changes of PFO such as straightening of the conjugated backbone and increase in conjugation length. Moreover, the non-solvent promotes intra-chain interactions such as p-stacking. To gain further insight into intramolecular interactions in PFO we performed photoluminescence experiments. Fig. 1c reveals a strong decrease in photoluminescence and a redshi upon addition of the non-solvent to the polyuorene. We propose a two-step model of PFO aggregation where aer addition of nonsolvent, in the rst few minutes stiffening of chains and an increase in conjugation length occurs that could be explained by a distinct redshi in absorption and photoluminescence spectra. Aer this initial phase, gradual quenching of photoluminescence occurs due to p-stacking which is leading to aggregates with photophysical features that resemble those usually explained in terms of J-aggregation model used for small molecules. [10][11][12][13] The next part of the study concerned the binding ability of both forms of PFO, dissolved and aggregated, to amyloid insulin brils. In particular, we employed UV-Vis absorption spectroscopy since binding of conjugated oligomers to amyloid brils is known to result in absorption shiing. Such an effect has been demonstrated previously for a number of polythiophene 5 and polyuorene derivatives 6,14 . Fig. 2 presents absorption and linear dichroism spectra of dissolved and aggregated PFO complexed with insulin brils. Both (i) well-dissolved PFO in ethanol and (ii) aggregated PFO are stabilized by the presence of amyloid insulin brils and no time dependent absorption shiing was observed. In contrast, PFO in pH ¼ 2 buffer alone displays a rapid redshi of the peak absorption to more than 400 nm (cf. Fig. 1b). Interestingly, when PFO aggregation was initiated through solvent exchange and the oligomer was allowed to aggregate for 20 min the addition of insulin brils promptly inhibited further spectral changes. These results indicate that insulin brils are able to stabilize the intermediate state of aggregation, as evidenced by the absence of any further redshi as well as the absence of any spectral changes (Fig. 2b). This observation is consistent with results obtained for dissolved PFO, for which attraction between the oligomer and amyloid brils is a driving force to form complexes. To investigate the structure of PFO and its interactions with insulin brils in more detail linear dichroism (LD) was used (Fig 2c). PFO in ethanol only displays weak LD signals but features a strong positive band when complexed with insulin brils at pH ¼ 2. The increase in LD amplitude is attributed to the rigidity and length of amyloid brils that orient better under ow. Hence, ow-aligned amyloid brils induce optical anisotropy in bound PFO. A positive LD signal suggests that PFO aligns with the oligomer backbone parallel to the long axis of the amyloid brils, which is consistent with previous reports. 15 The same orientation was also observed for PFO that was rst allowed to aggregate for 20 min followed by the addition of insulin brils, which arrest aggregation. Surprisingly, the LD spectrum features three distinct bands at 354 nm, 371 nm and 393 nm, indicating subtle structural changes in the oligomer backbone when compared with well dissolved PFO bound to brils. Similar effects were reported for small molecules that form J-aggregates. 16 We conclude that the observed spectral features are related to the aggregation rate where stiffening of chains and p-stacking interactions inuence the photophysics of the PFO oligomer. The LD, which provides information on the transition dipole moments in aligned molecules, is sensitive to structural changes that can occur during the aggregation process. Thus the spatial connement of the intermediate states of PFO aggregates on the bril surface enables control over the oligomer structure, which facilitates monitoring of the photophysical properties by absorption spectroscopy.
We conclude that attractive forces between amyloid brils and PFO outweigh any tendency for inter-or intrachain coupling of PFO that occur upon solvent exchange. The most plausible binding mode is that both forms of PFO, dissolved in ethanol and aggregated, align in the channels at the brils surface since interior binding sites are sterically hindered 17 and the orientation of the oligomer is driven by the rate of interactions with amyloid brils. Since there are no protonated functional groups, it is feasible that supramolecular interactions occur due to p-p stacking between aromatic residues and poly-uorene rings, which is maximized by the planar alignment of PFO at the surface. Ethylene glycol side chains may represent a further stabilizing factor, since they possess the ability to form hydrogen bonds with amino acids 18 in between protolaments of the brils and act as anchors. Since the aggregates are promptly stabilized by insulin brils it can be deduced that wellordered b-sheet structures act as nanotemplates for aggregating oligomers in solution. The presented results may be interpreted within the HJ-aggregation 10 model, which seems to be most appropriate for describing amyloid-PFO system.
Inter-and intramolecular interactions that occur during aggregation of PFO as well as complexation with amyloid brils can lead to cooperative effects that tend to strongly inuence the nonlinear absorption properties. 19 In order to investigate whether such effects can be observed in case of PFO aggregates stabilized on amyloid brils, we used the open-aperture Z-scan technique, which can be employed to determine the nonlinear absorption coefficient (b) (for details see Experimental section and ESI in ref. 20). The Z-scan technique is well-suited for determining TPA parameters in weakly emissive systems such as oligomer aggregates 21 or amyloid protein brils 20 , where methods based on measurements of TPA induced emission may not be suitable. Thus, we used this setup to explore whether PFO and its aggregates display an enhanced TPA signal when complexed with amyloid brils. Z-scans were performed at 800 nm, which is a suitable compromise wavelength for studying TPA of dissolved and aggregated PFO that one-photon absorption maximum is around 400 nm. Moreover, in this region amyloid brils themselves absorb only through a three-photon process, 20 which is a several orders of magnitude weaker effect and thus did not noticeably inuence the nonlinear absorption properties of PFO at 800 nm. Fig. 3a shows open aperture (OA) Z-scan traces for PFO in ethanol and theoretical ts that were produced by a custom program that used equations derived by Sheik-Bahae et al. 22 A detectable TPA signal was recorded for PFO and the nonlinear absorption strength was quantied as the value of the two-photon cross section s 2 , given in GM units where 1 GM ¼ 10 À50 cm 4 s photon À1 . For the PFO stock solution of 2 g L À1 the calculated TPA is s 2 ¼ 280 GM per monomer unit. However, in order to measure a TPA that is attributed only to conformational changes of PFO and furthermore to complexation with brils, the detection threshold for PFO in ethanol was determined by lowering the oligomer concentration to 0.5 g L À1 where no OA signal was recorded. The same concentration was used for further experiments where in a rst step PFO aggregates were formed by keeping oligomer in acidic water buffer for 20 min. Since aggregates are unstable without the presence of amyloid brils but precipitate, it was not feasible to record a corresponding two-photon absorption spectrum. Instead, the solution becomes turbid and strong laser light rapidly overheats the sample at the focal spot, which results in a sharp peak decrease in an OA measurement (Fig. S5 †). Similar experiments were performed with insulin brils, which stabilizes both the dissolved and aggregated form of PFO. Fig. 3b shows open aperture (OA) Z-scan traces for diluted PFO in dissolved and aggregated form bound to brils. Some uncertainty can arise from aggregate scattering since the theoretical t does not ideally match the experimental trace. Nevertheless, we observe a pronounced difference between data for dissolved and aggregated PFO attached to the brils surface. For the dissolved PFO bound to insulin brils the calculated TPA was s 2 ¼ 350 GM whereas a signicantly stronger signal for aggregated PFO-insulin brils complexes yielded a four times higher value of s 2 ¼ 1380 GM. This observation indicates that straightening of the conjugated backbone and stiffening of chains due to binding with amyloids leads to enhanced and sizable TPA, which can be attributed to through-bond cooperative effects. In case of aggregated PFO bound to insulin brils, through-space cooperativity can lead to further substantial enhancement of TPA signal. Thus both effects can contribute to the observed increase in TPA.
Reported values indicate that the use of conjugated polymers is advantageous as compared to standard chromophores such as ThT or Congo red in terms of two-photon absorption cross section, which is an important factor with regard to imaging techniques based on nonlinear optics. Whereas it is known that p-conjugation leads to a sizable increase by through-bond interactions, here we show for the rst time that upon aggregation additional enhancement occurs due to a close proximity of aggregated p-conjugated polymer on the bril surface. Compared to small molecules used as amyloid staining agents where the two-photon absorption cross section is $50 GM, aggregated polyuorene shows more than an order of magnitude higher values reaching nearly 1400 GM.

Conclusions
In summary, photophysical and optical properties of a poly-uorene derivative (PFO) and its binding to amyloid insulin brils were examined. Complexation based on weak supramolecular interactions between amyloid brils and PFO in dissolved and aggregated form led to sizable enhancement of twophoton absorption. Amyloid brils were used as nanotemplates that facilitate studies of nonlinear absorption cooperative effects in conjugated oligomers by stabilizing the intermediate states of aggregating molecules in solution.