Moleculer Motion in Aggregates: Manipulating TICT for Boosting

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Moleculer Motion in Aggregates: Manipulating TICT for Boosting Photothermal Theranostics Shunjie Liu, Xin Zhou, Haoke Zhang, Hanlin Ou, Jacky W. Y. Lam, Yang Liu, Linqi Shi, Dan Ding, and Ben Zhong Tang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13889 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 13, 2019

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Journal of the American Chemical Society

Shunjie Liu,†,# Xin Zhou,ǁ,‡# Haoke Zhang,† Hanlin Ou,‡,┴ Jacky W. Y. Lam,† Yang Liu,┴ Linqi Shi,*,┴ Dan Ding,*,‡,┴,§ Ben Zhong Tang*,†,§ †

Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Division of Life Science and State Key Laboratory of Molecular Neuroscience, and Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China ǁDepartment of Medical Imaging, Shanxi Medical University, Taiyuan 030001, China ‡ Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai University, Tianjin 300071, China ┴ State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials of Ministry of Education, and College of Chemistry, Nankai University, Tianjin 300071, China § Center for Aggregation-Induced Emission, SCUT-HKUST Joint Research Institute, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, 510640, China ABSTRACT: Planar donor and acceptor (D-A) conjugated structures are generally believed as the standard for architecting highly efficient photothermal theranostic agents, in order to restrict intramolecular motions in aggregates (nanoparticles). However, other channels of extra nonradiative decay may be blocked. Now this challenge is addressed by proposing an “abnormal” strategy based on: molecular motion in aggregates. Molecular rotors and bulky alkyl chains are grafted to the central D-A core to lower intermolecular interaction. The enhanced molecular motion favors the formation of dark twisted intramolecular charge transfer state, whose nonradiative decay enhances the photothermal properties. Result shows that small molecule NIRb14 with long alkyl chains branched at second carbon exhibits enhanced photothermal properties than NIRb6 with short branched chains and much higher than NIR6 with short linear chains and the commercial gold nanorods. Both in vitro and in vivo experiments demonstrate that NIRb14 nanoparticles can be used as nano-agent for photoacoustic imaging-guided photothermal therapy. Moreover, charge reversal poly(β-amino ester) makes NIRb14 specifically accumulate at tumor site. This study thus provides an excited molecular motion approach towards efficient phototheranostic agents.

The past few decades have witnessed significant efforts in the development of photothermal theranostics in terms of photoacoustic (PA) imaging and photothermal therapy (PTT).1-8 Among the existing photothermal agents, near-infrared (NIR)absorbing organic dyes and polymers have received considerable attention owing to their good biocompatibility, potential biodegradability, easy processability, and high reproducibility.9-16 In particular, organic dye/polymer nanoparticles (NPs) using biocompatible amphiphilic co-polymers as the doping matrix are extremely valuable, since the matrix usually endow the organic dyes/polymers with excellent colloidal stability, desirable blood circulation time, passive tumor targeting ability by the enhanced permeability and retention (EPR) effect, and flexible tumor microenvironment responsiveness.17-21 Thereby, one of the most challenging issues is that the PA imaging and PTT efficacies of the organic dye/polymer NPs are often limited by their low photothermal properties. As most currently reported organic dyes and semiconducting polymers simply constructing strong donor (D) and acceptor (A) units into coplanar structures, the resulting strong intermolecular interactions among the molecules inside the NPs cores significantly block other channels

of heat generation.11 This leads to finite photothermal conversion efficiency and tremendous difficulty for further improvement of photothermal property. To address the challenge, many contributions have focused on exploring new approaches to largely boost the photothermal properties of organic dye/polymer NPs. For instance, Lee and coworkers increased the photothermal properties by attaching a light-harvesting unit into the semiconducting polymer.22 Pu and co-workers reported an intraparticle photoinduced electron transfer (PET) method to quench the fluorescence of NIRabsorbing semiconducting polymers by co-encapsulation of electron-withdrawing fullerenes into NPs.23 The amplified nonradiative decay by intraparticle PET results in enhanced PA imaging and PTT efficacies. These intelligent methods open up new avenues for designing more efficient PA imaging/PTT used semiconducting polymer NPs. However, the essence is still based on planar structures of the conjugated polymers, and the complexity in polymer structure hinders the clear mechanistic study. Moreover, incorporation of the additional component into NPs decidedly compromises the preparation reproducibility, which is not conducive to clinical translation. Therefore, it

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is urgent to develop a new, simple yet clear strategy to significantly boost the photothermal properties of organic dye/polymer NPs. Recently, an excited state electron transfer process observed in small molecules, termed twisted intramolecular charge transfer (TICT), has received our attention.24-26 In this process, after photoexcitation, the dark TICT state returns to the ground state mainly through nonradiative relaxation, accompanying redshifted emission (Scheme S1). Notably, the prerequisite to the formation of TICT state relies on active molecular rotations. In the TICT process, the susceptibility of this state favors various nonradiative quenching process, thus better TICT character could favor enhanced heat generation.24-25 Molecular systems based on TICT mechanism are well known and reported in the literature;24-26 however, their use for biological/biomedical applications has been rarely investigated.27 On the other hand, in 2001, our group coined the concept of aggregation-induced emission (AIE), in which the dye molecules are non-emissive in solution but become highly fluorescent in aggregates owing to the mechanism of restriction of intramolecular motion.28-30 These two concepts seem to conflict to each other in aggregates, because the dark TICT state is forbidden by the constricted molecular motions, while AIE luminogens (AIEgens) are highly emissive in this case. However, the emission of AIEgens can be gradually quenched by adding good solvent to the aggregates (reverse AIE process), due to the activation of molecular motions. Based on these considerations, we propose a strategy based on: “Molecular motion in aggregates”, combining the advantages of dark TICT and reversed AIE into one method. Accordingly, improved photothermal conversion can be achieved owing to the stabilization of dark TICT state and restriction of fluorescence decay.

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Herein, as a proof-of-concept, we introduced molecular rotors into planar D-A based NIR-absorbing small organic molecule, which is helpful for both molecular motion and stabilization of the dark TICT state. Moreover, besides further increasing TICT property, long alkyl chains were used as shielding units to restrain intermolecular interactions, and most importantly to maintain the intramolecular motions in aggregates.31 Specifically, we designed and synthesized D-A conjugated small molecules based on low-bandgap benzo[1,2-c:4,5c′]bis([1,2,5]thiadiazole) (BBTD) as acceptor, thiophene as πconjugation unit and donor, triphenylamine (TPA) as molecular rotor and meanwhile the second donor, and long alkyl chains branched at the second carbon as the shielding units. It is found that the molecule NIRb14 (Figure 1A) with long 2-decylmyristyl group displayed much boosted photothermal conversion property, PA effect over short branched NIRb6 and NIR6 with short 1-hexyl chain and the widely used gold nanorods (GNR). Furthermore, to prolong in vivo blood circulation time and enhance tumor accumulation and retention, poly(β-amino ester)b-poly(caprolactone) (PAE-b-PCL) and PEG-b-PCL were used to formulate NIRb14 into mixed-shell NPs, since PAE can respond to tumor acidic microenvironment.32 Live mouse study verifies that our proposed concept based on "adjusting TICT in aggregates for boosting photothermal property" together with the help of PAE-based NPs enable NIRb14 NPs a far superior PA imaging agent to commercial dye indocyanine green (ICG), which also show excellent in vivo PTT efficacy with reduction of tumor volumes. Collectively, this study offers a new molecular guideline to design advanced photothermal theranostic agents through manipulation of TICT.

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NIR6 Figure 1. Molecular design of NIRb14, NIRb10, NIRb6 and NIR6 for PA imaging-guided PTT. (A) Chemical structure, (B) schematic illustration of TICT state in solution, and (C) aggregation state, (D) the scheme of the NIRb14 and NIR6 NPs for PA imaging-guided PTT.


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Synthesis and Characterization. The D-A conjugated small molecules were synthesized by using BBTD as a strong acceptor, which possesses a substantial quinoidal character within a conjugated backbone, allowing for greater electron delocalization and thus lowering of the band gap;33-37 and thiophene as both donor and π-conjugation unit (Figure 1A, Scheme S2, S3). This strong electron-deficient structure can promote the non-radiative decay and in turn quench the fluorescence. The twisted TPA unit acts as the molecular rotors to assure the intramolecular rotation; meanwhile, as the second donor group to construct D’-D-A-D-D’ type dye. The planar thiophene ring in between can facilitate the intramolecular charge transfer (ICT) from TPA to BBTD.38 Alkyl chains located at the thiophene bridge are introduced to tune the molecular structures and states. Molecule grafted with branched long 2-decylmyristyl group is denoted as NIRb14, less longer 2-octyldecyl as NIRb10, short 2ethylhexyl as NIRb6, while linear 1-hexyl unit as NIR6 (Figure 1A), and their structures associated intermediates are well characterized by 1H and 13C NMR as well as time of flight mass spectra (Figure S1-S20). The key structural feature of NIRb14 is the second-position branched alkyl chains on the thiophene, as it provides suitable steric hindrance that prevents the aggregation of the molecule too strong like a linear 1-hexyl group or causes too much hindrance resulting in fluorescence like alkyl chains branched at first carbon.39 In addition, the alkyl chain engineering can be conducted by molecules NIRb6, NIRb10 and NIRb14, with progressively increased chain length. Moreover, molecule NIR6 with linear hexyl group is also designed to compare the steric effect with NIRb6 (branched hexyl unit). Accordingly, in contrast to NIR6, NIRb14 possesses larger steric hindrance and longer alkyl chains. The alkyl chain does not influence the fluorophore unit in a direct way (like through a conjugated effect or Förster resonance energy transfer), but indirect via a slight change of the environment.26 It is noticeable that the long alkyl chains extended out of the conjugate backbone can regulate the TICT state in solution (Figure 1B), since the ICT rate constant increases with the length of alkyl chains.40 Furthermore, in aggregates, the long side chains in NIRb14 can act as shielding unit to facilitate intramolecular rotation to promote the formation of the dark TICT state (Figure 1C), because molecular rotation plays a crucial role in TICT state.26, 41 While, in NIR6, the intermolecular interactions dominate, which restricts the intramolecular motions. In addition, the introduction of long alkyl chains gives the final compound improved solubility in common solvents like, tetrahydrofuran (THF), dichloromethane (DCM), chloroform, et al., and also higher synthetic yield (Supporting Information). The energy gap between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of all the molecules is about 1.2 eV (Figure 2). These narrow band gaps are beneficial for intense absorption in the NIR biological window.42 On the other hand, the molecular HOMO is delocalized along the whole molecular backbone, while the LUMO is more localized on the central D-A-D core. Expectedly, the central D-A-D core is planar, while there is a large distortion from the planar structure between central core and TPA unit. Thus, the spatial separation of HOMO and LUMO are favorable to the formation of TICT state.43 Notably, the gradually increased dihedral between BBTD and thiophene from NIR6 to NIRb14 indicates that branching and lengthening of alkyl chains are beneficial for backbone twisting. Collectively, compared with NIR6, the better TICT property of NIRb14 particularly in aggregate state

within NPs is expected to enable higher photothermal properties and thus better photothermal theranosis effect in vivo (Figure 1D). HOMO

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TICT property. There are many microenvironmental factors which influence the behavior of dyes in the excited state and thus affect the location and/or intensity of the observed emission in the photoluminescence (PL) spectra.44 The photophysical properties of all the molecules were conducted. As shown in Figure 3A, there are two strong absorption bands for the molecules in THF, which are assigned to a high-energy ππ* transition and a low-energy charge-transfer (CT) band.45-46 The invariable high-energy π-π* band (360 nm) could be possibly attributed to the planar D-A-D backbone.47 NIRb14 displays better CT ability over the other three molecules, as reflected by their CT absorption band increases in an order of 808, 813, 819 and 822 nm for NIR6, NIRb6, NIRb10 and NIRb14, respectively, again demonstrating a more twisted structure imposed by a long and branched alkyl chain. Obviously, the maximum absorption wavelengths of these four molecules are in the first NIR range (NIR-I; ca. 700–900 nm), which can penetrate much deeper tissue, and cause less photodamage to live organisms.38 To compare their TICT properties, the solvatochromic effect was studied.48-49 As shown in Figure 3B, the maximum emission wavelength (λem) of the molecules increases with solvent polarity. For example, the λem increases from 1030 to 1115 nm and from 1016 to 1094 nm by progressively increasing solvent polarity from toluene (PhMe) to dimethylformamide (DMF) for NIRb14 and NIR6, respectively, while accompanying a decreased PL intensity. Generally, molecules with branched side chains display red-shifted emission compared to NIR6 with linear ones. Moreover, in branched chain series, NIRb14 with longest alkyl chains shows the largest Stokes shift, again suggests a notable change in geometry in the TICT state.26 To more clearly study the TICT property, the effects of solvent polarity on the emission are evaluated quantitatively by Lip2∆𝑓 (𝜇𝐺 − 𝜇𝐸 )2 + pert−Mataga equation: 𝜈̃𝐴 − 𝜈̃𝐸 = ℎ𝑐𝑎3 𝑐𝑜𝑛𝑠𝑡,50-51 and the experimental data are summarized in Table


Journal of the American Chemical Society S1 and Figure S21. 𝜈̃𝐴 and 𝜈̃𝐸 represent the maximum absorption and emission wavenumber, h is Planck’s constant, c is the speed of light and a is the Onsager radius of the solute spherical cavity. The physical variable µG represents the dipole moment of the ground state and 𝜇𝐸 is the dipole moment of the excited state. The solvent polarity parameter ∆𝑓 can be calculated by

difference between the ground and the excited state dipole moment ∆µ which can be calculated from the slope of the linear regression. As shown in Figure 3C, the slopes of the molecules from NIRb14 to NIRb6 are 6572, 6326 and 6060 cm -1, respectively, which are higher than NIR6 of 5197 cm-1. The relatively high slope in NIRb14 suggests a highly polarized excited state.26

𝑛2 −1

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the following equation: ∆𝑓 = 𝑓(ε) − 𝑓(𝑛2 ) ≈ − 2 , 2𝜀+1 2𝑛 +1 using the dielectric constant ε and refractive index n of the solvent. A plot of the Stokes shifts (𝜈̃𝐴 − 𝜈̃𝐸 ) versus ∆𝑓 yields the

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Figure 3. Optical and photothermal properties of NIRb14, NIRb10, NIRb6 and NIR6. (A) Normalized absorption spectra in THF. (B) Maximum emission wavelength (λem) in different solvents(C) Correlation of solvent polarity parameter with Stokes shift. (D) Variation of PL intensity (I/I0) with water fraction in THF/water mixtures. (E) Change in PL intensity with DMSO fraction in DMF/DMSO mixtures. (F) Relative QY measured in THF and water. (G) Powder XRD spectra. (H) Comparison of the photothermal conversion behavior of the molecules NPs in PBS solution at the same concentration (100 μM) with GNR. (I) Photothermal conversion ability of the NIRb14 NPs with different dye/PEG-b-PCL mass ratio at the same dye concentration (100 μM). The 808 nm laser (0.8 W/cm2) was irradiated for 5 min, then the laser was removed, and the samples were naturally cooling down to ambient temperature.

To prove the TICT character in aggregates, water (bad solvent) was systematically added to a THF solution of these dyes. With the increase of water content in THF (fw), the PL signals of the molecules decrease progressively (Figure 3D), because the strong solvent relaxation in the excited state results in the conversion of ICT to dark TICT.26 Notably, at fw = 90%, the emission of NIRb14 is completely quenched rather than NIR6,

whose fluorescence can still be detected. This result indicates that the shielding unit in NIRb14 can protect the TICT state from the intermolecular interactions. Whereas, for NIR6, the strong intermolecular interaction hinders the formation of dark TICT state and the fluorescence appears, as evidenced by recent studies that intermolecular interactions can enhance fluorescence once aggregates form.52 Moreover, at fw = 40%, NIRb14


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displays rather low PL intensity ratio (I/I0) of 24% over NIR6 (I/I0= 57%), while a relative larger emission maximum (1083 vs 1064 nm) is observed (Figure S22). These results once again demonstrate that NIRb14 is superior to NIR6 for the stabilization of the dark TICT state. To study the intermolecular interactions effect in aggregates, we judiciously choose a strong polar solvent mixture of DMF and dimethyl sulfoxide (DMSO) with similar polarity, because these molecules are weakly soluble in DMF but insoluble in DMSO. The weak and broad emissions of these two molecules in DMF indicates the large-amplitude relaxation in the dark TICT state, which mainly weaken the fluorescent emissions via non-radiative decay.53 As shown in Figure 3E and S23, at DMSO fraction of 90%, the I/I0 of the molecules from NIRb14 to NIR6 is 5%, 18%, 61% and 72%, respectively. These results further prove that the long alkyl chain in NIRb14 can facilitate intramolecular rotation in aggregates, thus activates the nonradiative decay. However, the existence of strong intermolecular interactions in NIR6 or even NIRb6 restricts the intramolecular motion, and thus restores fluorescence via a radiative pathway. A similar phenomenon is also observed in water, NIR6 NPs (PEG-b-PCL as the doping matrix) displays relatively stronger PL intensity, whereas, the emission of NIRb14 NPs is too weak to exhibit normal distribution (Figure S24). Moreover, NIRb14 displays the lowest relative quantum yield (QY) both in THF and water (NPs), again demonstrating its better TICT property (Figure 3F). Furthermore, from the powder X-ray diffraction spectrum in Figure 3G, NIRb14 shows obviously increased interplanar crystal spacing over NIR6 according to Bragg’s equation, which is beneficial for molecular motions. Collectively, the present strategy enhances the TICT properties of the molecules in engineering more useful aggregate state (nanoparticle).

PCL as the doping matrix by a nanoprecipitation method, 32, 54 we next evaluated the photothermal properties of the resulting NPs. Gold-based nanoparticles have manifested extraordinary promise for PTT,55-59 therefore, PEG coated gold nanorod (GNR) was used as a control.4 Quantitative measurements of the temperature changes of these NPs are depicted in Figure 3H. In NIRb14 NPs, their aqueous solution temperature reaches a maximum at 78 °C with a 54 °C temperature increase (∆T) after 808 nm laser irradiation (0.8 W/cm2) for 5 min, which is higher than NIRb10 NPs (∆T ~ 51 °C) and NIRb6 (∆T ~ 48 °C) and is much higher than NIR6 NPs (∆T ~ 45 °C) and GNR (∆T ~ 31 °C). The higher maximum photothermal temperature and better photothermal conversion of NIRb14 NPs than NIR6 NPs are attributed to the longer alkyl chains with a suitable branched site that permit both intramolecular motion and stabilization of dark TICT state in aggregate state within NPs. To study whether the alkyl chains existed in PEG-b-PCL can affect the photothermal properties of dye, we formulated NIRb14 into different amount of PEG-b-PCL. As shown in Figure 3I, the maximum photothermal temperature was independent of PEG-b-PCL amount. Also, the photothermal properties of these NPs were not affected by their dynamic light scattering (DLS) sizes (Figure S25, S26). Further, control experiments using DMF/H 2O (1/9, v/v) and pure DMF solution all demonstrated that the superb photothermal properties of NIRb14 over NIR6 and commercial dyes GNR and ICG (Figure S27). Thus, the superb photothermal properties of NIRb14 were resulted from its better TICT property and the molecular motion in aggregates.

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Photothermal Property. Upon formulation of the four molecules into NPs, respectively, using co-polymer PEG-b-

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It can be intuitively visualized that NIRb14 NPs exhibit a clear NIR-photothermal effect upon exposure to 808 nm laser for 5 min (Figure 4A). Additionally, the photothermal effect is



(control NPs with only PEG5k-b-PCL5k as the matrix) remains negative in the pH range from 5.0 to 7.4. Moreover, the PAEencapsulated NPs display excellent long-term colloidal stability and good cytocompatibility (Figure S32-S33). In addition, the highly boosted tumor cellular uptake of NIRb14-PAE/PEG NPs at pH of 6.5 validates their efficient charge conversion (Figure S34). It is noted that the mean size of NIRb14-PAE/PEG NPs at pH 6.5 negligibly alters when compared with that at pH 7.4 measured by DLS (Figure S35), indicating that the surface charge reversal does not influence the NP stability in aqueous media. These results demonstrate that PAE endows NIRb14PAE/PEG NPs with excellent charge conversion feature in response to tumor acidic microenvironment. In vivo PA Imaging of NIRb14-PAE/PEG NPs. Before PA imaging in live animals, the in vitro PA property of NIRb14PAE/PEG NPs was studied. As shown in Figure 6A, the NIRb14-PAE/PEG and NIRb14-PEG NPs have almost identical PA maximum at ~808 nm, which is in good agreement with absorption profile of NIRb14 molecule (Figure 2A). In addition, the PA performance is not affected by different PAE loading or simply assembled in DMF/H2O mixtures (Figure S36). Thus, we can prove that the presence of PAE hardly affects PA performance. The linear relationship between PA intensity with the concentration of the four molecules indicates the great potential of these NPs in quantitative PA imaging (Figure 6B). Notably, NIRb14-PAE/PEG NPs possess far better PA performance in contrast to the other three molecules and organic ICG, which also show ~1.5 times higher PA amplitude than NIR6PAE/PEG NPs at a dye concentration of 100 μM, verifying the advantage of our concept "adjusting TICT in aggregates for boosting photothermal property". B

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positively related to the NIRb14 NPs concentration (Figure 4B). Noteworthy, the NIRb14 NPs exhibit outstanding photostability, without losing maximum absorption after 30 min of NIR irradiation, in marked contrast, nearly complete elimination of NIR absorbance in commercial ICG is observed (Figure S28). Furthermore, even after 5 cycles of heating and cooling, NIRb14 NPs can still recover the maximum temperature to the original level (79 °C) as that of NIR6 NPs and GNR in contrast to ICG, suggesting far stronger resistance to photobleaching (Figure 4C, S29). In addition, NIRb14 NPs display excellent colloidal stability even after 5 cycles of photothermal tests (Figure S30). According to the method published in the literatures,60-62 the photothermal conversion efficiency (PCE) at 808 nm of NIRb14 NPs in aqueous solution is determined to be 31.2%, which is higher than the NPs of NIRb10 (29.8%), NIRb6 (26.2%), NIR6 (22.6%) and GNR (20.7%) (Figure S31). This result indicates that the PCE of the molecules increases with chain length, however, the rate of increase is decreased when alkyl chain length increased to some extent (2-decylmyristyl unit). As a consequence, the strong and stable photothermal performance of NIRb14 makes it an encouraging agent for in vivo PTT and PA imaging. NPs Charge Conversion via Tumor-Acidity Responsiveness. To obtain prolonged blood circulation and increased tumor uptake and retention in vivo, a pH-responsive polymer, poly(β-amino ester)-b-poly(caprolactone) (PAE-b-PCL),63-64 was employed as an additional co-polymer to prepare NPs. PAE possesses rapid and reversible pH-responsive character, which is protonated and hydrophilic at tumor acidic microenvironment (pHe ~6.5), while it can be deprotonated and hydrophobic at physiological pH (~7.4). As shown in Figure 5A, both amphiphilic co-polymers PEG5k-b-PCL5k and PAE7k-b-PCL5k with a weight ratio of PEG5k to PAE7k segment of 1:1 are utilized to encapsulate NIRb14 molecules via the nanoprecipitation method at pH = 4, since at this pH, PAE7k segment is watersoluble. The afforded NIRb14-encapsulated PAE7k-bPCL5k/PEG5k-b-PCL5k NPs (named NIRb14-PAE/PEG NPs for short) has a hydrophobic core consisting of entangled PCL and NIRb14 molecules as well as a mixed shell of both hydrophilic PEG and PAE at pH = 4. Upon tuning the pH to 7.4 that mimics the physiological pH in blood, the PAE segments in the mixed shell become deprotonated, leading to the formation of the hydrophobic surface domain by the collapse of hydrophobic PAE, which enable the micro-phase separation on the surface of NPs to occur (~4.91 × 104 NIRb14 molecules per nanoparticle). It has been well established that such micro-phase separation on the NPs surface greatly benefits to prolonged blood circulation time in vivo due to the reduced opsonin adsorption.32 DLS and transmission electron microscopy (TEM) observations indicate that NIRb14-PAE/PEG NPs have a nearly spherical shape with a hydrodynamic diameter of ~134 nm (Figure 5B). On the other hand, at tumor microenvironment with pH ~6.5, the protonated PAE7k chains extend to the surrounding water and the positively charged PAE7k could decidedly enhance the tumor cellular uptake and tumor retention time of the NIRb14PAE/PEG NPs (Figure 5A), since in general the cell membranes are negatively charged.65-69 To confirm the charge conversion property of the NIRb14-PAE/PEG NPs in response to acidic tumor environment, the zeta potential measurement was carried out. As displayed in Figure 5C, the NIRb14-PAE/PEG NPs exhibit distinct charge conversion at pH of 6.8 and positive signal increases from -2.9 to +9.5 eV with decreasing the pH from 7.4 to 5.0. As a control, the zeta potential of NIRb14-PEG NPs

NIRb14 -PAE/PEG NPs

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Figure 6. (A) PA spectra in PBS solution. (B) PA amplitudes at 800 nm as a function of dye NPs concentration. (C) Transverse section views of PA images at tumor sites from mice before (0 h) and after intravenous injection of NIRb14-PAE/PEG or NIRb14-PEG NPs (660 μM based on NIRb14) at designated time intervals. The region in the red dotted half-ellipse indicates the tumor location. (D) PA intensity at tumor site as a function of time postinjection of NIRb14-PAE/PEG and NIRb14-PEG NPs, respectively. Error bars, mean ± s.d. (n = 3). *** P < 0.001 in comparison between the two NPs groups.


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Saline

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We further conduct the in vivo PA tumor imaging by intravenous injection of NIRb14-PAE/PEG NPs into xenograft 4T1 tumor-bearing mice and using NIRb14-PEG NPs as a control. Figure 6C exhibits the PA images of the tumors before (0 h) and after intravenous administration of NIRb14-PAE/PEG and NIRb14-PEG NPs, respectively. As shown in Figure 6D, at 7 h post-injection, the PA signals of NIRb14-PEG NPs at the tumor sites reach to the plateau, while the plateau time is at 9 h for NIRb14-PAE/PEG NPs. From the linear relationship between PA intensity and dye concentration, we can calculate the concentration of NIRb14 molecule in tumor region at 9 h was 44.09 ± 4.55 µM and the %ID/g was 6.84±0.85%. The result indicates the better tumor accumulation and retention of PAE-based NPs. This should be ascribed to the unique role of PAE on the NPs, that at pH 7.4 in blood, micro-phase separation on the surface results in prolonged blood circulation and hence enhanced EPR effect, whereas, at tumor acidic pH, the reversed positive charges in PAE chains significantly favor the cell uptake and retention of NPs within tumors. In addition, post 96 h injection, the PA signals drop to almost the initial level, implying the facile catabolization of NPs in vivo.70 Imaging-Guided Phototherapy In Vivo. Motivated by the attractive PA imaging ability, the PA image-guided PTT capability of the NIRb14-PAE/PEG NPs was investigated in vivo using xenograft 4T1 tumor mouse model. Tumor-bearing mice were randomly divided into 6 groups, which were named as “Saline”, “NIRb14-PEG NPs”, “NIRb14-PAE/PEG NPs”, “Saline + laser”, “NIRb14-PEG NPs + laser”, and “NIRb14PAE/PEG NPs + laser”, respectively. For the first three groups, saline, NIRb14-PEG NPs, and NIRb14-PAE/PEG NPs were intravenously injected into the mice in the corresponding cohort. For the other three groups, at 7 h post intravenous injection of each agent, the tumors of mice in each group were continuously irradiated with an 808 nm laser for 5 min.

C

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Figure 7. (A) IR thermal images of 4T1 tumor-bearing mice under 808 nm laser irradiation (0.8 W/cm2) for different times. The laser exposure was performed at 7 h after intravenous injection of 150

μL of NIRb14-PAE/PEG NPs, NIRb14-PEG NPs, and saline, respectively. [NIRb14-PAE/PEG NPs] = [NIRb14-PEG NPs] = 660 μM based on NIRb14 (B) The temperature changes at the tumor sites as a function of the 808 nm laser (0.8 W/cm2) irradiating time, which is the quantitative data of (A). Error bars, mean ± s.d. (n = 4). (C) Tumor growth curves of mice in different treatment groups. Error bars, mean ±s.d. (n = 8). * P < 0.05 in comparison between "NIRb14-PAE/PEG NPs + laser" and "NIRb14-PEG NPs + laser" groups.

To verify the heat generation capacity of our NPs in living mice, after intravenous injection of each NPs or saline for 7 h, the tumor temperatures of mice in each group were monitored over time by IR thermography upon exposure to 808 nm laser irradiation. As shown in Figure 7A and 7B, negligible temperature variation (∆T ~ 4°C) is observed from the tumors in “Saline + laser” group. Noteworthy, tumors from mice in “NIRb14PEG NPs + laser” cohort exhibit a temperature elevation of about 21°C. Encouragingly, much higher temperature rise (∆T ~ 29 °C) with maximum temperature to 64 °C is observed from the tumors in “NIRb14-PAE/PEG NPs + laser”-treated mice. As PAE has negligible interference in the photothermal conversion property of NIRb14, such higher photothermal temperature at the tumor mass is attributed to the better tumor accumulation and retention of NPs by virtue of the surface PAE with tumor acidic microenvironment responsiveness signature. The in vivo antitumor efficacies of the aforementioned treatments were examined by monitoring the tumor volumes for 16 days (the treatments were performed on day 0). As depicted in Figure 7C, the treatments of NPs without 808 nm laser irradiation, like “NIRb14-PEG NPs” and “NIRb14-PAE/PEG NPs” fail to suppress tumor growth (average tumor volume increasing by 12-fold), similar to those of control groups (“Saline” and “Saline + laser”). The result suggests that pure 808 nm laser exposure or NPs themselves without laser have a negligible activity for cancer therapy. On the other hand, the “NIRb14-PEG NPs + laser” treatment via PTT exhibits excellent antitumor efficacy with tumor increase in volume only by 0.8-fold on day 16. The most remarkable antitumor efficacy comes from the “NIRb14-PAE/PEG NPs + laser” group, whose average tumor volume on day 16 is even smaller than day 0. Such highly effective tumor growth suppression should be ascribed to not only the excellent photothermal activity of NIRb14, but also the prominent tumor uptake ability of PAEcaped NPs. In addition, the almost invariable body weight loss in mice from these 6 groups suggests the low side toxic effect of NIRb14 and its NPs (Figure S37). The therapeutic effect of each treatment was also assessed at the microscope level. In this experiment, the mice in all six groups were sacrificed and the tumors were excised for hematoxylin and eosin (H&E) staining as well as immunohistochemical analyses including Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) (Figure S38).38 H&E staining clearly indicates that the treatment of “NIRb14-PAE/PEG NPs + laser” causes much more necrotic areas at tumor site. Furthermore, both TUNEL and PCNA staining demonstrate that “NIRb14PAE/PEG NPs + laser” treatment is the most efficacious in inducing the apoptosis and suppressing the proliferation capacity of tumor cells. It is also worthy to note that negligible tissue damage and inflammatory lesion are observed in the reticuloendothelial system (RES) organs such as liver and spleen, manifesting the harmlessness of NIRb14-PAE/PEG NPs to these important normal organs (Figure S39).19 Furthermore, the



intravenously injected NPs can be nearly completely cleared from mice body after 9 days via biliary pathway (Figure S40 and S41).

In summary, we have put forward a new excited molecular motion approach: “adjusting TICT in aggregates for boosting photothermal property” to highly enhance the photothermal properties for PA imaging and PTT of tumors in live mice. The introduction of the molecular rotor (TPA) and 2-decylmyristyl bulky groups into the planar D-A-D core is beneficial for molecular motion and stabilization of dark TICT state. Importantly, the integration of long alkyl chain (branched at second carbon) is of vital importance. The enhanced TICT property on the basis of breaking the intermolecular interaction in the aggregate state within NPs results in NIRb14 showing much better photothermal conversion capacity and higher photothermal temperature elevation than short branched NIRb6 and linear NIR6 and widely used GNR. Furthermore, the mixed-shell NPs formulation method by employing tumor-acidity-responsive PAE endows NIRb14-doped NPs with prolonged blood circulation, enhanced EPR effect, and improved tumor retention, hence achieving superb in vivo PTT efficacy as evidenced by the significant shrinkage of tumors at the end of the study. To the best of our knowledge, this study reports for the first time that utilizing dark TICT state can promote the photothermal conversion property and PA effect, which also provides one of the very few examples of associating microcosmic molecular motion with biomedical function and effectiveness.

Supporting Information. General information about materials and methods, synthesis and characterizations, NMR spectra of the compounds.

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected].

Shunjie Liu: 0000-0003-1632-0183 Dan Ding: 0000-0003-1873-6510 Ben Zhong Tang: 0000-0002-0293-964X #S.

L. and X. Z. contributed equally to this work.

The authors declare no competing financial interest.

We are grateful for financial support from the National Science Foundation of China (51622305, 21788102, 21490570, 21490574 and 51873092), the National Key Research and Development program of China (2018YFE0190200), the Research Grants Council of Hong Kong (16308116, 16308016, C2014-15G, C6009-17G, NHKUST604/14 and A-HKUST605/16), the Innovation and Technology Commission (ITC-CNERC14SC01, ITCPD/17-9 and ITS/254/17), the National Basic Research Program of China (2015CB856503), and the Science and Technology Plan of Shenzhen (JCYJ20160229205601482 and JCY20170818113602462).

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Journal of the American Chemical Society TICT state

Aggregation state

NIR

PA

heat NIR

PA heat

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Figure 1. Molecular design of NIRb14, NIRb10, NIRb6 and NIR6 for PA imaging-guided PTT. (A) Chemical structure, (B) schematic illustration of TICT state in solution, and (C) aggregation state, (D) the scheme of the NIRb14 and NIR6 NPs for PA imaging-guided PTT. 311x191mm (150 x 150 DPI)


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Figure 2. Calculated HOMOs, LUMOs, and optimized ground-state (S0) geometries of the molecules. 263x244mm (150 x 150 DPI)



Figure 3. Optical and photothermal properties of NIRb14, NIRb10, NIRb6 and NIR6. (A) Normalized absorption spectra in THF. (B) Maximum emission wavelength (λem) in different solvents(C) Correlation of solvent polarity parameter with Stokes shift. (D) Varia-tion of PL intensity (I/I0) with water fraction in THF/water mixtures. (E) Change in PL intensity with DMSO fraction in DMF/DMSO mixtures. (F) Relative QY measured in THF and water. (G) Powder XRD spectra. (H) Comparison of the photothermal conversion behavior of the molecules NPs in PBS solution at the same concentration (100 μM) with GNR. (I) Photothermal conver-sion ability of the NIRb14 NPs with different dye/PEG-b-PCL mass ratio at the same dye concentration (100 μM). The 808 nm laser (0.8 W/cm2) was irradiated for 5 min, then the laser was removed, and the samples were naturally cooling down to ambient tempera-ture. 151x144mm (150 x 150 DPI)


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Figure 4. (A) IR thermal images of the NIRb14 NPs (100 μM) and GNR under 808 nm laser irradiation (0.8 W/cm2) for differ-ent times. (B) Photothermal conversion behavior of NIRb14 NPs at different concentrations (5−100 μM) under 808 nm laser irradiation. (C) Antiphotobleaching property of NIRb14 NPs, NIR6 NPs and GNR (100 μM based on the dye molecule) dur-ing five circles of heating−cooling processes. 720x656mm (150 x 150 DPI)



Figure 5. (A) Schematic design of pH-responsive NIRb14-PAE/PEG NPs. (B) DLS profile and TEM image (inset) of the NIRb14-PAE/PEG NPs at pH 7.4. (C) Zeta potential changes of NIRb14-PAE/PEG NPs and NIRb14-PEG NPs incubated in buffers at different pH values at 37 °C. 860x1020mm (150 x 150 DPI)


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Figure 6. (A) PA spectra in PBS solution. (B) PA amplitudes at 800 nm as a function of dye NPs concentration. (C) Transverse section views of PA images at tumor sites from mice before (0 h) and after intravenous injection of NIRb14-PAE/PEG or NIRb14-PEG NPs (660 μM based on NIRb14) at designated time intervals. The region in the red dotted half-ellipse indi-cates the tumor location. (D) PA intensity at tumor site as a function of time postinjection of NIRb14-PAE/PEG and NIRb14-PEG NPs, respectively. Error bars, mean ± s.d. (n = 3). *** P < 0.001 in comparison between the two NPs groups. 210x184mm (150 x 150 DPI)



Figure 7. (A) IR thermal images of 4T1 tumor-bearing mice under 808 nm laser irradiation (0.8 W/cm2) for different times. The laser exposure was performed at 7 h after intravenous injec-tion of 150 μL of NIRb14PAE/PEG NPs, NIRb14-PEG NPs, and saline, respectively. [NIRb14-PAE/PEG NPs] = [NIRb14-PEG NPs] = 660 μM based on NIRb14 (B) The temperature changes at the tumor sites as a function of the 808 nm laser (0.8 W/cm2) irradiating time, which is the quantitative data of (A). Error bars, mean ± s.d. (n = 4). (C) Tumor growth curves of mice in different treatment groups. Error bars, mean ±s.d. (n = 8). * P < 0.05 in comparison between "NIRb14-PAE/PEG NPs + laser" and "NIRb14-PEG NPs + laser" groups. 170x185mm (150 x 150 DPI)


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Moleculer Motion in Aggregates: Manipulating TICT for Boosting

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