Combining Two-Photon-Activated Fluorescence Resonance Energy

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Combining Two-Photon Activated Fluorescence Resonance Energy Transfer and Near Infrared Photothermal Effect of Unimolecular Micelles for Enhanced Photodynamic Therapy Yu Huang, Feng Qiu, Lingyue Shen, Dong Chen, Yue Su, Chao Yang, Bo Li, Deyue Yan, and Xinyuan Zhu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b06450 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

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Combining Two-Photon Activated Fluorescence Resonance Energy Transfer and Near Infrared Photothermal Effect of Unimolecular Micelles for Enhanced Photodynamic Therapy Yu Huang,† Feng Qiu,*,‡ Lingyue Shen,# Dong Chen,† Yue Su,† Chao Yang,§ Bo Li,§ Deyue Yan,*,† and Xinyuan Zhu*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix

Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People‟s Republic of China. ‡

School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100

Haiquan Road, Shanghai 201418, People‟s Republic of China. #

Department of Oral Maxillofacial-Head Neck Oncology, Ninth People‟s Hospital, Shanghai

Jiao Tong University, 639 Zhizaoju Road, Shanghai 200011, People‟s Republic of China. §

Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal

University, 500 Dongchuan Road, Shanghai 200241, People‟s Republic of China. * Corresponding author: X. Y. Zhu ([email protected]) or F. Qiu ([email protected]) or D. Y. Yan ([email protected]).

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ABSTRACT Recent years have witnessed significant progress in the field of two-photon activated photodynamic therapy (2P-PDT). However, the traditional photosensitizer (PS) based 2P-PDT remains a critical challenge in clinics due to its low two-photon absorption (2PA) cross-sections. Here, we propose that the therapeutic activity of current PSs can be enhanced through a combination of two-photon excited fluorescence resonance energy transfer (FRET) strategy and photothermal effect of near infrared (NIR) light. A core-shell unimolecular micelle with large two-photon absorbing conjugated polymer core and thermo-responsive shell was constructed as a high two-photon light-harvesting material. After grafting PSs onto the surface of unimolecular micelle, the FRET process from conjugated core to PSs could be readily switched „„on‟‟ for killing cancer by the collapsed thermo-responsive shell due to the photothermal effect of NIR light. Such NIR-triggered FRET leads to an enhanced 2PA activity of the traditional PSs, and in turn amplifies their cytotoxic singlet oxygen generation. Eventually, both in vitro and in vivo PDT efficiency treated with the thermo-responsive micelles were dramatically enhanced under NIR light irradiation, as compared to pure PSs excited by traditional visible light. Such a facile and simple methodology for the enhancement of the photodynamic antitumor effect holds great promises for cancer therapy by further development. KEYWORDS two-photon absorption, photothermal effect, NIR, unimolecular micelle, thermoresponsiveness, photodynamic therapy


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Photodynamic therapy (PDT) is a promising systemic treatment for cancer because of its noninvasive selective destruction.1,2 In PDT, the photosensitizers (PSs) play a major role, which can generate cytotoxic species, e.g. singlet oxygen (1O2), after excitation in the diseased tissues.3,4 Recently, the two-photon activated photodynamic therapy (2P-PDT) using near infrared (NIR) light excitation offers a perspective due to its potentials for deeper penetration and much greater precision in treatment as compared to the traditional PDT.5,6 Unfortunately, the clinical applications of 2P-PDT are limited by the low two-photon absorption (2PA) cross-sections (1~50 GM, 1 GM = 10-50 cm4 s/photon) of readily available PSs.7,8 Even though continuous efforts have been made to the design of PSs with increased 2PA activity, some disadvantages of their complicacy and time-consuming still exist, especially the ignoble 1O2 generation capability due to the insufficient triplet formation.9 Alternatively, 2PA molecules with large cross-sections as an energy up-converting donor for exciting the current PSs through a two-photon activated fluorescence resonance energy transfer (2P-FRET) strategy would be an attractive and practical approach to realize an effective 2P-PDT.10 To date, the co-encapsulation of 2PA molecules and PSs has been widely employed to prepare the 2P-PDT platform.11-13 However, the emission performance is commonly deteriorated by the inherent π-π interaction from the conjugated 2PA molecules and hydrophobic PSs in their agglomerate state, leading to an unsatisfactory FRET effect.14,15 In addition, the efficiency of FRET is closely related to the distance between 2PA molecules and PSs. Thus, design of a 2PA system with enhanced FRET process to PSs is crucial to the PDT therapeutic efficacy. As a proof-of-concept, incorporation of a stimuli-responsive polymer with controllable phase transition between 2PA molecules and PSs would be a highly desirable to achieve the 2P emission enhancement and 2P-FRET amplification. Up to now, various stimuli-responsive


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polymers with controlled phase transition of coil-globule responded to various stimuli, such as pH, light, and temperature have been explored extensively during the past decade.16-19 Among them, thermal-responsive polymers are a popular candidate, since the reversible phase transition could be controlled easily and conveniently by local heating.20 Benefiting from the high sensibility, phase transition from extended coil to collapsed globule in thermo-responsive polymers would happen instantaneously when the temperature is up to the lower critical solution temperature (LCST).21 We also noted that the occurrence of photothermal effect with temperature enhancement is generally accompanied with the irradiation of NIR light, which is surprisingly neglected before.22 Therefore, it can be inferred that the 2P-PDT could be realized simply via 2P-FRET strategy under the irradiation of NIR light source, if a temperatureresponsive polymer with the LCST around body temperature is built to couple the 2PA molecule and PS. Based on this concept, a core-shell unimolecular micelle composed of a 2PA hyperbranched conjugated polymer (HCP) core and thermo-responsive hyperbranched polyether (HPE) shell, named as HCP@HPE, was designed as a 2P light-harvesting material. With the protection of compact HPE shell outside the conjugated core, excellent emission properties of HCP@HPE including one-photon (1P) and 2P emission could be achieved in the aqueous medium. Fortunately, we succeeded in applying the NIR light to switch „„on‟‟ and „„off‟‟ the FRET process from HCP to Chlorin e6 (Ce6, a commercial PS) for amplifying 1O2 generation after grafting Ce6 onto the surface of HCP@HPE. In vitro and in vivo results clearly demonstrated that our thermo-responsive nanoplatform provided a remarkable enhancement of tumor inhibition induced by the combination of 2P-FRET and photothermal effect of NIR, as compared to free Ce6 and traditional visible light PDT.


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RESULTS AND DISCUSSION Synthesis, Optical Properties and Self-Assembled Behavior of HCP@HPE-Ce6. As shown in Figure 1, the HCP core with large 2PA cross-sections and hydroxyl terminal groups was prepared according to our previous work.23,24 Subsequently, HPEs were grafted onto HCPCH2OH by oxyanionic proton-transfer polymerization with trimethylolpropane (TMP), trimethylolethane (TME), 1,4-butanediol diglycidyl ether (BDE), and potassium hydride (KH, as the catalyst) to form core-shell unimolecular [email protected],26 Here, three kinds of HCP@HPE samples with different molar ratios of BDE to various polyols were prepared (Table S-1), named as HCP@HPE1, HCP@HPE2 and HCP@HPE3. Their detailed characterizations of HCP@HPE are displayed in Figures S1-S3. Benefiting from the hydrophilic HPE shell, the resultant HCP@HPE could dissolve well in water. As an example of HCP@HPE3, Figure 2a shows a set of UV-vis absorption bands at 308 and 346 nm, which is the typical π-π* transition of 3,6carbazole units and phenyleneethylene moieties, respectively. Upon excitation at 365 nm, HCP@HPE3 shows blue luminescence with maximum emission (λem) at 445 nm. Utilizing the chromophore of 9,10-diphenylanthracene (quantum yield = 90.0%) as a reference probe, the quantum yield of 26.83 ± 0.13% and emission lifetime of 4.47 ± 0.14 ns were acquired from HCP@HPE3 in Table S-2. The photophysical results were summarized in Table S-3. By employing an 800 nm femtosecond laser (iHR550, HORIBA) as the excitation, HCP@HPE3 shows strong two-photon fluorescence (2PF) at 453 nm (Figure 2b). The 2PF intensity increases with the square of the light intensity, and the corresponding average 2PA cross-section is 1335 ± 177 GM (Rhodamine-B as the reference).27 Compared with those of HCP in THF solvent (Figure S5), the optical properties of HCP@HPE in water display weak red-shift, suggesting a slight effect of HPE shells on HCP core. Moreover, the existence of HPE shells in HCP@HPE micelles


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could restrict the intermolecular aggregation of HCP core through a mechanism of „„multimicelle aggregates (MMA)‟‟,28-30 resulting in good emission performance of HCP@HPE in aggregation state. To clarify its self-assembled behavior, both dynamic light scattering (DLS) and transmission electron microscopy (TEM) were utilized to studied the size and morphology of HCP@HPE micelles in water. DLS result gives bimodal distribution (Figure 2c), in which the small one at 13.7 ± 1.5 nm is assigned to the unimolecular micelles of HCP@HPE, consistent with its dimension calculated by Chem3D (Figure S10);31 while the large one at 85.6 ± 5.9 nm comes from the self-assembled MMA. TEM results also reveal the coexistence of two different sizes of HCP@HPE nanoparticles (Figures S8 and S9), further confirming the formation of MMA from its unimolecular micelles.30 The illustration of self-assembly mechanism of amphiphilic HCP@HPE is demonstrated in Figure S11. HPE is a classical thermo-responsive polymer with LCST property, and the phase transition of HCP@HPE aqueous solution could be measured by temperature dependent turbidimetry.25 Figure 2d shows the LCST of HCP@HPE3 is 39.1 ± 0.1 oC. Firstly, the HCP@HPE3 aqueous solution is transparent at room temperature. As the temperature is increased by heating, the solution gradually becomes opaque, then it turns back to transparent again when the temperature is cooled down. This temperature-responsive behavior is attributed to the combined interaction between hydrophilic segments (-OH and -O-) and hydrophobic segments (-CH3, -C4H8- and CH2CH3) in the structure of HPE shells. When the temperature is up to LCST, the interreaction of hydrophobic segments will actuate backbones to collapse and then aggregate in aqueous solution. Besides, with the decrease of hydrophilic polyols, the LCST value of correspondent HCP@HPE decreases almost linearly (Figure S7). These data suggest that LCST value of HCP@HPE can be easily adjusted to body temperature for potential bioapplications. In the


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present work, HCP@HPE3 with LCST value at 39.1 ± 0.1 oC is chosen for the further study of photodynamic antitumor effect. Owing to the abundant hydroxyl terminal groups on the surface of HCP@HPE3, Ce6 can be easily grafted by esterification reaction, named as HCP@HPE3-Ce6. The detailed preparation information, Ce6 grafting content and corresponding characterizations are given in Figures S4 and S12-S14. As expected, Ce6 shows UV-vis absorption peaks at 404, 510 and 656 nm, which overlaps well with fluorescence emission spectrum of HCP core (Figure S6). Thus, the FRET process will be enhanced efficiently just when the distance between HCP and Ce6 is shortened. Figure 2e gives the temperature dependent fluorescence spectra of HCP@HPE3-Ce6, in which emission peaks at 445 nm and 682 nm attribute to HCP (IHCP) and Ce6 (ICe6), respectively. When the temperature is below 39 oC, the emission at 682 nm changes a little; once the temperature is above 39 °C, the intensity at 682 nm is increased dramatically. The intensity ratios of ICe6/IHCP as a function of temperature are adopted to describe the change of FRET process. As shown in Figure 2f, the ICe6/IHCP of HCP@HPE3-Ce6 is dramatically improved when the temperature is above LCST, indicating that enhanced FRET of HCP@HPE3-Ce6 can be realized by the temperature-induced collapse from HPE shells. These results are able to correlate the variabletemperature UV-vis observation. Upon the 2P excitation at 800 nm, similar results are obtained by compared with that of 1P excitation, demonstrating the occurrence of 2P-FRET in HCP@HPE3-Ce6 micelles over the LCST of 39 oC (Figure S14i). When the temperature reaches 40 oC, the fluorescence intensity of Ce6 at 682 nm increases remarkably with the emission quenching of HCP at 445 nm. In addition, the reversible change of ICe6/IHCP can be controlled repeatedly through heating or cooling due to the reversible phase transition of HPE shells (Figure S14j).


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Cellular Uptake of HCP@HPE-Ce6. Based on the importance of subcellular distribution of PSs for the PDT effect, the study of cellular uptake to pure Ce6 (Ce6 sodiumsalts) and HCP@HPE3-Ce6 micelles was evaluated by flow cytometry and confocal laser scanning microscopy (CLSM). As shown in Figure S15a and S15b, the mean fluorescent intensity of HeLa cell treated with HCP@HPE3-Ce6 micelles during the first 0.5 h incubation is a little higher than that of free Ce6-pretreated cells. This observation shows a higher cellular uptake from HCP@HPE3-Ce6 micelles due to the consumption of negatively charged carboxyl groups in the Ce6 moieties, as compared to free Ce6.32 With incubating time prolonging, the fluorescence intensity increases linearly (Figure S15c). These obvious fluorescent signals are just related to the attachment of HCP@HPE3-Ce6 micelles in the HeLa cells. Furthermore, the samples could be directly observed by CLSM (Figures S16 and S17). For HCP@HPE3-Ce6 micelles, both fluorescence from HCP and Ce6 are observed mainly in the cytoplasmic regions of cells from blue and red tunnels respectively, indicating the successful internalization by living cells. The fluorescence intensity of cell imaging is also enhanced as the increase of incubating time, which is in good agreement with those of flow cytometry results. 2P-FRET Behavior and TLSM Imaging of HCP@HPE-Ce6 in Living Cells. The good cellular uptake results infer us to verify the 2P-FRET behavior of HCP@HPE3-Ce6 micelles in the cellular environment via two-photon laser scanning fluorescence microscopy (TLSM, Nikon A1R). For the fixed HeLa cells, a strong blue fluorescent signal from HCP is viewed in the cytoplasm upon the excitation at 800 nm (Figure S19a); while no red signal of Ce6 is detected. It suggests the higher 2PA performance of HCP than that of Ce6. After continuous irradiation for 15 min, little blue fluorescence is localized in the cells, and the red fluorescence of Ce6 is enhanced dramatically (Figure S19b), which is attributed to the “switching on” 2P-FRET process


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from HCP to Ce6 through the NIR irradiation. To verify the photothermal effect of NIR, we monitored the temperature of cellular environment under the irradiation of NIR light (Figure S18). After 15 min irradiation, the temperature would reach 39.8 ± 0.1 °C in the cells, which is higher than LCST value of HCP@HPE3, leading to the collapse of HPE. Thus, the enhanced emission of Ce6 indeed results from the efficient 2P-FRET. The whole time-dependent FRET process in fixed cells by 2P excitation is given in Figure S20. In addition, these dynamic processes could also be observed in living cells under the 800 nm irradiation (Figure 3a). The red fluorescence of Ce6 increases visibly with the quenching of HCP blue fluorescence, revealing the progressive enhancement of 2P-FRET of HCP@HPE3-Ce6 micelles in living cells. Meanwhile, after irradiation with 800 nm laser up to 15 min, the HeLa cells show low survivability, owing to the effective activation of Ce6 for cell killing. All these results confirm that the activation of Ce6 by use of 2P-FRET process is workable in the living cells. In Vitro 2P-PDT Effect. The in vitro dark-cytotoxicity of free Ce6, HCP@HPE3 and HCP@HPE3-Ce6 was studied by using MTT assay on HeLa cancer cells. Up to a concentration of 100 μmol/L, cell viabilities treated by free Ce6, HCP@HPE3 and HCP@HPE3-Ce6 after 24 h of incubation remain 84.5 ± 1.7%, 82.8 ± 2.3% and 83.6 ± 1.9% in the absence of irradiation respectively, in contrast to the untreated cells (Figure S21). All the results demonstrate that these materials show low dark-cytotoxicity on HeLa cells without irradiation of light. In vitro photocytotoxicity of free Ce6 and HCP@HPE3-Ce6 was evaluated by 2P laser irradiation at 800 nm for 20 min (Figure 3b). As a control, the test was performed under the 1P laser irradiation of 650 nm, which is a typical direct excitation of the Ce6. Owing to the fluence-rate-dependent photocytotoxicity of Ce6,33 the intensities of 650 nm were fixed at 12 J/cm2; 2P laser power at 800 nm (3 scans of 1 s) was set at 100% and focused by a microscope objective lens (10×/0.3), and the


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mean energy per pulse was 0.7 mJ. Under 650 nm laser irradiation at 37 oC, the dose of Ce6 needed for 50% inhibition of cell growth (IC50 value) is 15.17 ±0.35 and 12.32 ± 0.29 μg/mL for free Ce6 and HCP@HPE3-Ce6 micelles, respectively. The lower dose required for micelles is ascribed to the higher cellular uptake of HCP@HPE3-Ce6 micelles than that of negative charged Ce6 (as previously cellular uptake results displayed). Upon 800 nm laser irradiation at 37 oC, both free Ce6 and HCP@HPE3-Ce6 micelles show low inhibition proliferation of HeLa cells. The primary reason is that Ce6 possesses the small 2PA cross-sections under direct 2P excitation. For micelles, the low 2P-FRET happens below LCST. Once the temperature reaches 40 oC after 800 nm irradiation, the distance between HCP and Ce6 is shortened by the collapse from HPE shells, which switches “on” the 2P-FRET of HCP@HPE3-Ce6 micelles, leading to an efficient activation of Ce6 for killing cells. Consequently, the inhibition of HeLa cells is enhanced greatly by the HCP@HPE3-Ce6 micelles treatment. The IC50 value of HCP@HPE3-Ce6 falls to 3.95 ± 0.21 μg/mL, which is just a quarter of dosage in contrast to traditional Ce6 treatment (1P excitation at 650 nm). These results demonstrate that 2P-FRET process is an effective approach for killing the cancer cells in photodynamic treatment, which has been rarely reported in the literature.34 The reason might be the molar extinction coefficient of Ce6 at 450 nm is higher than that at 650 nm, resulting in a higher cytotoxic 1O2 generation. To further confirm the aforementioned in vitro PDT efficacy, the direct 1O2 generation capability of various formulations is evaluated by oxygen luminescence spectra. In Figure 3c, the peak at 1270 nm, attributing to the characteristic 1O2 emission,35 is observed clearly in the free Ce6 deuterated solution with visible light (650 nm) excitation at 37 oC; while HCP@HPE3-Ce6 deuterated solution shows the similar intensity at 1270 nm, due to the same concentration of Ce6. Under the same condition, both free Ce6 and HCP@HPE3-Ce6 show the low 1O2 generation after


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the excitation at 800 nm. Differently, a remarkable enhancement of 1O2 emission from deuterated HCP@HPE3-Ce6 solution is found when the temperature rises to 40 oC under the excitation at 800 nm, indicating the high 1O2 generation capability. These results are consistent with those of in vitro photo-cytotoxicity. The more 1O2 is generated from activation of Ce6, the lower dosage of Ce6 is needed. To quantitatively determine 1O2 generation from Ce6 units under different condition, we further used the 9,10-anthracene-dipropionicacid (ADPA) as a 1O2 quencher,13 which could react with 1O2 and lead to the decrease of its optical absorption at 378 nm. Figure 3d gives the relationship between the absorption at 378 nm of an ADPA solution bearing free Ce6 or HCP@HPE3-Ce6 and the irradiation time. For the group pretreated by HCP@HPE3-Ce6 micelles in the temperature above LCST at 40 oC, a significant decrease of the absorption at 378 nm is clearly observed after the irradiation at 800 nm in contrast to others. It reflects that the high 1

O2 content has been produced from HCP@HPE3-Ce6 micelles via a 2P-FRET strategy. As a

consequence, the viability of HeLa cells with the treatment of HCP@HPE3-Ce6 micelles, upon the irradiation of low cytotoxic 2P laser at 800 nm (Figure 3e), is decreased greater than others with the light of prolonged in Figure 3f, demonstrating that the heating process generated by NIR light irradiation is efficient to switch on the 2P-FRET for PDT, which agrees well with the TLSM results discussed previously in the living cells. In general, apoptosis is the dominant mechanism of tumor death in PDT. Thus, two apoptosis indicators: caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were selected to further elucidate cytotoxicity. In order to study whether caspase-3 proteins were up-regulated by 2P-PDT or not, Ac-DEVD-pNA was utilized as a substrate to detect the protein expression of caspase-3. HeLa cells were pretreated with PBS, free Ce6 and HCP@HPE3-Ce6 micelles, respectively, at 20 μg/mL of Ce6 for 4 h. Figure 3g demonstrates that


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the caspase-3 protein expression gives a bit of enhancement against HeLa cells in the group of PBS (with 650 nm and 800 nm irradiation treatment), free Ce6 (with non-laser, 650 nm and 800 nm irradiation treatment), and HCP@HPE3-Ce6 micelles (with non-laser and 650 nm irradiation treatments) in comparison with the control of PBS (non-laser). Differently, The remarkable upregulated expression of caspase-3 are observed in HeLa cells treated with HCP@HPE3-Ce6 micelles upon 800nm irradiation. Besides, the apoptosis-induced DNA fragmentation was revealed by TUNEL (green fluorescence signal) (Figure S22). It can be seen that no green fluorescence signal is observed via a green tunnel after the treatment of PBS, free Ce6 (nonlaser), and HCP@HPE3-Ce6 micelles (non-laser); while a little bit of green fluorescence is emitted in the free Ce6 (650 nm and 800 nm irradiation) and HCP@HPE3-Ce6 micelles (650 nm irradiation) pretreated cells. In particular, the green staining of TUNEL is positive observably after the treatment of HCP@HPE3-Ce6 micelles at 800 nm irradiation for 15 min, which reveals a severe cell apoptosis. Thus all the observations indicate that the apoptosis of cancer cells induced by the PDT effect could be enhanced efficiently through a 2P-FRET strategy, which is in compliance with the previous PDT activity in vitro. Pharmacokinetics and Biodistribution Assessments. The pharmacokinetic evaluation was conducted by use of Sprague-Dawley (SD) rats with intravenous injection of free Ce6 and HCP@HPE3-Ce6 micelles (0.10 mmol Ce6 kg-1). Figure 4a gives the content variation of free Ce6 and HCP@HPE3-Ce6 micelles as a function of the time in serum. It can be seen that HCP@HPE3-Ce6 micelles exhibit improved pharmacokinetic property as compared to free Ce6, ascribing to nano-characteristics of micelles, which suggests a high tumor accumulation ability of HCP@HPE3-Ce6 micelles. Furthermore, the distribution of HCP@HPE-Ce6 micelles in vivo, especially in tumor region, was tracked through Ce6-NIR fluorescence imaging on the whole


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body.36 After injecting free Ce6 and HCP@HPE-Ce6 micelles intravenously into the mice containing HeLa tumor via tail vein, the real-time animal imaging of free Ce6 and HCP@HPECe6 micelles was monitored during the period of 12 h post-injection. As shown in Figure 4b, fluorescence of Ce6 mainly distributes in organs outside the tumor after injection of free Ce6; while the fluorescence of Ce6 from HCP@HPE-Ce6 micelles pre-injected mice gradually increases in the tumor region over the whole time; even at 12 h after the injection, the signal remains detectable. The largest accumulation of HCP@HPE-Ce6 micelles in the tumor is about 4 h after administration. At the different time interval of 1, 4 and 8 h time points, the ex vivo NIR fluorescence imaging of excised tumors and various organs was used to determine the definite distribution of drug (Figure 4c). For free Ce6, the fluorescence signal is only visible in the liver, and little signal can be detected in the tumor. Similar to that of whole animal imaging, a clear signal of Ce6 units in the tumor could be visualized by injection of HCP@HPE-Ce6 micelles, especially at point of 4 h. These imaging results illustrate that nano-micellized HCP@HPE-Ce6 would greatly increase the stability of Ce6 in the blood circulation and promote the selective accumulation of Ce6 in tumors. To determinate the content of Ce6 in various organs via intravenous injection of free Ce6 and HCP@HPE-Ce6 micelles, the mice were sacrificed at different pre-injection time, and the Ce6 levels in all collected tumors and organs were measured by fluorescence spectroscopy (Figure 4d). Similar to the aforementioned biodistribution results, the concentration of Ce6 from HCP@HPE-Ce6 micelle-treated groups in the tumors and organs is remarkably higher than that of free Ce6 groups. All these data indicate that HCP@HPE-Ce6 micelles can provide relatively long retention time and possess high tumor accumulation via the enhanced permeability and retention (EPR) effect. Importantly, it should be mentioned that the enrichment of HCP@HPE-Ce6 micelles in the tumor reaches a maximum accumulation at about


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4 h post-injection, which guides that 4 h after administration is the optimal therapeutic time point by PDT process. In Vivo Antitumor Activity Studies of HCP@HPE-Ce6 Micelles. Being aware of the high NIR-induced 1O2 generation capability, high 1O2-mediated apoptosis activity, and the excellent tumor accumulation effect of HCP@HPE-Ce6 micelles, we finally carried out in vivo 2P-PDT treatment studies using HeLa tumor-bearing nude mice.12,13 The mice were randomly separated into nine groups (Figure S23, n = 5) and were intravenously injected with 200 μL PBS, free Ce6, and HCP@HPE-Ce6 micelles solution at doses of Ce6 0.10 mmol/kg, respectively. At 4 h postinjection, the tumor regions were then irradiated at three different areas of the tumor separately with 2P-800 nm and 1P-650 nm laser for 0.5 h, and the groups without light exposure were used as controls. Body weight monitoring is one of indicators to evaluate therapeutic effect. As shown in Figure 5a, a slight drop in body weight is observed for the PBS and free Ce6 (with non-laser, 650 nm and 800 nm irradiation) treated groups because of the negative effects from the low therapeutic efficacy. However, with respect to the treatment of HCP@HPE-Ce6 micelles at 800 nm laser irradiation, a little increase in body weight is detected, which suggests a steady body growth after the effective therapy during 20 days. Meanwhile, the tumor volumes (Figure 5b) treated with HCP@HPE-Ce6 micelles at 800 nm laser irradiation are much smaller than that treated with PBS (with non-laser, 650 nm and 800 nm irradiation), free Ce6 (with non-laser, 650 nm and 800 nm irradiation), and HCP@HPE-Ce6 micelles (non-laser and 650 nm irradiation). Significantly, the evident tumor necrosis can be viewed in the experiment group treated with HCP@HPE-Ce6 micelles (800 nm laser) in the 10th day (Figure S23, red arrow labelled), demonstrating an effective treatment of HCP@HPE-Ce6 micelles via the 2P-FRET strategy. After 20 days post-treatment, tumor growths are remarkably suppressed by the treatment of


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HCP@HPE-Ce6 micelles at 800 nm laser exposure (Figure5c). The tumor inhibitory rate (TIR) is counted with tumor weight in Figure 5d. In contrast to the PBS without laser irradiation, the TIR to HCP@HPE-Ce6 micelles (800 nm irradiation) is 87.1 ± 1.7%, which is significantly higher than other groups. These results declare that the 2P-PDT efficacy of HCP@HPE-Ce6 micelles is the highest one among all therapeutic groups. The antitumor efficacy after treatment with various formulations was further evaluated by histologic analyses.37 In Figure 6a, the staining of hematoxylin and eosin (H&E) from tumor biopsy pretreated with PBS (non-laser, 650 nm laser, or 800 nm laser) shows that large nucleus and spindle shape are observed due to the rapid tumor growth. The similar results are achieved in the mice after intravenous injection with free Ce6 (non-laser and 800 nm irradiation treatment) and HCP@HPE-Ce6 micelles (non-laser treatment). By contrast, the nucleus shrinkage and fragmentation are reviewed after treatment with free Ce6 (650 nm irradiation) and HCP@HPECe6 micelles (650 nm irradiation), implying that the inhibition of tumor cells is gained by the traditional visible light PDT. However, for the mice pretreated HCP@HPE-Ce6 micelles under irradiation at 800 nm, most tumor cells are severely destroyed in tissue slice, which reveals that high cell apoptosis is activated by 2P-PDT effect of HCP@HPE-Ce6 micelles. Furthermore, we also utilized the TUNEL and caspase-3 staining to study the photodynamic antitumor effect in different experimental groups. As expected in Figure 6b and 6c, almost none of tumor tissues can be stained in the PBS groups. In regard to free Ce6, a little bit of positive cells after TUNEL (positive: green) and caspase-3 (positive: brown) staining are displayed in the group preilluminated with 650 nm laser. Noteworthy, we find numerous apoptotic cells in the group receiving HCP@HPE-Ce6 micelles and 800 nm irradiation. As a result, a strong destruction of tumor tissue (around 90% suppression) is obtained by the HCP@HPE-Ce6 based 2P-PDT


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process (see red arrow in Figure 6d). All experimental results of H&E, TUNEL, caspase-3 staining and tumor inhibition confirm the superior in vivo 2P-PDT efficacy of HCP@HPE-Ce6 micelles, which is consistent with the previous in vitro PDT effect. In addition, no mortality was observed for the 20 days following the injection of HCP@HPE-Ce6 micelles, suggesting that these nanoparticles are not toxic except in the tumor area. Furthermore, we also studied the potential toxic side effect of HCP@HPE-Ce6 micelles based 2P-PDT process. H&E staining of major organs after 20 days treatment doesn’t reveal noticeable abnormal damages in comparison with the controls (before injection) (Figure S24). Combining with the MTT results in vitro darkcytotoxicity above, all of data suggest that our HCP@HPE-Ce6 micelle has no obvious toxicity, which proves that the 2P-PDT based on our micelles can be used as a safe and efficient antitumor technique. Currently, 2P-PDT, which offers deeper penetration, less photo-damage and more confined treatment areas than traditional visible light PDT, has emerged as one important tool in cancer therapy. Even though such excellent optical behaviors in NIR regions, an effective 2P-PDT based on the traditional PSs remains a critical challenge, owing to its limited 2P absorption. As a solution, 2PA molecules as an energy up-converting donor for indirectly exciting the PSs through a 2P-FRET strategy would be an attractive approach to enhance 2P-PDT activity of the current PSs. To date, plenty of conjugated polymers (CPs) with huge 2PA cross-sections value have been widely exploited as a 2P light-harvesting material. However, due to the serious π-π stacking of conjugated CPs and PSs, the fluorescence emission is not satisfying. To avoid this aggregation-induced quenching effect, the CPs and PSs should be isolated by subject. Considering that the FRET is closely related to the distance from donor to acceptor,


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incorporation of a stimuli-responsive polymer with reversible stretch-to-collapse property between CPs and PSs would be highly desirable for optimizing the FRET process. CONCLUSIONS To

summarize,

a

core-shell

unimolecular

hyperbranched@hyperbranched

micelle

(HCP@HPE) containing a 2P fluorescent HCP core and thermo-responsive HPE shell was synthesized via oxyanionic proton-transfer polymerization, and the commercialized PS of Ce6 was grafted on the shell of HCP@HPE by esterification reaction. We firstly report to use lightto-heat transduction effect accompanied with NIR light irradiation to switch „„on‟‟ the 2P-FRET from CPs to PSs for photodynamic antitumor therapy. Under irradiation at NIR light, the local temperature would be heated over than LCST of HCP@HPE, leading to the distance between HCP and PS shortened by the collapse of HPE shell. In NIR-fluorescence imaging, the fluorescence of PSs of HCP@HPE-Ce6 increases under the excitation at 800nm along with the fluorescence quenching of HCP, which is beneficial to generate cytotoxic 1O2 in PDT implement. More significantly, the concentration of 1O2 produced by HCP@HPE-Ce6 micelles under the irradiation at 800 nm via 2P-FRET strategy is much higher than that of free Ce6 treated with traditional 650 nm laser excitation. Consequently, the required Ce6 content of HCP@HPE-Ce6 for IC50 is the quarter of dosage used in traditional free Ce6 treatment. The in vitro and in vivo PDT results clearly showed that HCP@HPE-Ce6 micelles treated with NIR irradiation showed the highest PDT treatment efficacy among all therapeutic groups. Taking the advantage of high stability of in the blood circulation and selective accumulation in tumor, our nanoplatform holds great promises for enhancement of photodynamic antitumor therapy.


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MATERIALS AND METHODS Materials. HCP with hydroxyl terminal group (HCP-CH2OH) was prepared according to our previous work.23,24 Sodium borohydride (NaBH4, C.P. grade, Shanghai Chemical Reagent Co.), trimethylolpropane (TMP, Acros), trimethylolethane (TME, Acros), 1,4-butanediol diglycidyl ether (BDE, Aldrich), potassium hydride (KH, 30 wt% dispersion in mineral oil, Aldrich). N,N'Dicyclohexylcarbodiimide (DCC, 99%, J&K), N-hydroxysuccinimide (NHS, Acros), 4(dimethylamino)pyridine (DMAP, 99%, J&K), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) were directly utilized. N,N-Dimethylformamide (DMF), toluene, chloroform, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ethanol and dichloromethane were further dried before use. TUNEL assays were conducted with the TUNEL fluorescent kit from Beyotime in China. Casepase-3 antibody was from Cell Signaling Technology (#9662, USA). The secondary antibodies were obtained from Earthox LLC. (Earthox, USA). Preparations of HCP@HPE and HCP@HPE-Ce6. The HPEs were grafted onto HCPCH2OH to obtain HCP@HPE via oxyanionic proton-transfer polymerization on the basis of our previous paper25. Then, benefiting from the abundant hydroxyl terminal groups outside the HCP@HPE, the Ce6 can be easily grafted onto HCP@HPE by esterification reaction, named HCP@HPE-Ce6. Detailed materials' preparations, characterizations and experimental methods were deposited in the Supporting Information. Self-Assembly of HCP@HPE3-Ce6 Micelles. HCP@HPE3-Ce6 was dissolved in DMSO before use. In the process of stirring, water was added gradually into certain content of DMSO solution. HCP@HPE3-Ce6 nanoparticle aqueous solution was obtained by dialysis against


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ultrapure water for 48 hours. The final concentration of solution was adjusted by adding some distilled water. Cell Culture. HeLa cells were cultured in Dulbecco's Modified Eagle's medium (DMEM). The culture mediums contain 10% fetal bovine serum (FBS) and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. In vitro Photo-Cytotoxicity Effect. For the effect of photo-irradiated cell death, HeLa cells were firstly seeded into a multi-well plate. Incubated for 4 h after addition of free Ce6 and HCP@HPE3-Ce6 micelles, cells were washed twice, maintained in fresh culture medium, and then entire area of the well was photo-irradiated by a 650 nm laser (12 J/cm2, PDT650 Diode Laser Devices, Xing Da Photo & Electrics Medical Instrument Co., Ltd, China); and 2P laser at 800 nm (3 scans of 1 s), whose power was set at 100% and focused by a microscope objective lens (10×/0.3), and the mean energy per pulse was 0.7 mJ. After photo-irradiation, the cell viability was evaluated by MTT assay. Animals and Tumor Models. All Balb/c female nude mice (~20 g) and Sprague-Dawley rats (~200 g) were purchased from Chinese Academy of Sciences (Shanghai, China). Animal studies were conducted according to the guidelines of Animal Experimentation Ethics Committee proved by the Animal Ethics Committee of Shanghai Jiao Tong University. The female nude mice were injected subcutaneously with 200 μL of 4 × 106 HeLa cells per tumor. The tumors were allowed to grow to ~200 mm3 before experimentation. In Vivo Photodynamic Anticancer Studies. Mice were randomly separated into nine groups (n = 5) and were intravenously injected with 200 μL PBS, free Ce6, and HCP@HPE-Ce6


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micelles solution at doses of Ce6 0.10 mmol/kg, respectively. At 4 h post-injection, the tumor regions were then irradiated separately with 2P-800 nm and 1P-650 nm laser for 0.5 h at three different areas of the tumor, and the groups without light exposure were used as controls. The laser irradiation was performed with 1 min interval per 3 min light exposure. In our experiment, the tumor volume (V) and body weight were monitored every two days. V was counted with the formula: V (mm3) = 1/2 × length (mm) × width2 (mm2). After 20 days post-injection, all tumors were carefully harvested, weighted and photographed.

ASSOCIATED CONTENT Supporting Information Synthesis details of HCP@HPE and HCP@HPE-Ce6; 1H NMR, FTIR, UV-vis and fluorescence test results for the materials; self-assembly of HCP@HPE and HCP@HPE-Ce6 nanoparticles; DLS and TEM data of nanoparticles; Flow cytometry and cell apoptosis assay; singlet oxygen generation; CLSM and TLSM imaging; in vitro PDT experiment; animals and tumor models; pharmacokinetics and biodistribution; in vivo PDT experiment and immunohistochemical assessment. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected]


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Author contributions X. Z. and D. Y. planed and supervised the project. Y. H., F. Q. and X. Z. designed and carried out the detailed experiments, participated in result analysis and wrote the whole paper. Y. H. prepared the materials and carried out the spectroscopic measurements. Y. S., L. S. and D. C. assisted with the imaging and photodynamic therapy in vitro and in vivo. C.Y. and B. L. contributed to 2PA test and analysis. All authors reviewed the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work is supported by the National Basic Research Program of China (2015CB931801), National Key Research and Development Plan of China (2016YFA0201500), and National Natural Science Foundation of China (51473093, 21504057). We thank Dr. G. S. Tong from Instrumental Analysis Center of Shanghai Jiao Tong University for the TEM test of unimolecular micelles and multi-micelle aggregates. We also thank Dr. X. Gu and Dr. J. P. Ao for the help of TLSM imaging.


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FIGURES

Figure 1. Synthesis of thermo-responsive HCP@HPE unimolecular micelles and illustration of the combination of 2P-FRET and photothermal effect of NIR for photodynamic therapy.


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Figure 2. The optical properties and self-assembled behavior of HCP@HPE3 and HCP@HPE3Ce6. (a) Normalized absorption and PL spectrum of HCP@HPE3 in aqueous environment. (b) Normalized 2PF spectra of HCP@HPE3 aqueous solution under different laser intensity at 800 nm, Inset: the power dependence of 2PF intensity. (c) Typical intensity-weighted DLS plot of HCP@HPE3 particle in aqueous environment. (d) Temperature dependence of optical transmittance at 550 nm for HCP@HPE3 aqueous solution, Inset: digital photograph of HCP@HPE3 aqueous solution when heating and cooling. (e) Normalized PL spectra of HCP@HPE3-Ce6 aqueous solution with variable temperature at 25, 30, 35, 40, 42, 45, and 55 oC; (f) ICe6/IHCP values from HCP@HPE3-Ce6 as a function of temperature.


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Figure 3. TLSM imaging, PDT effect, 1O2 generation capability and the apoptosis activity of HCP@HPE3-Ce6 in vitro. (a) Dynamic FRET process of HCP@HPE3-Ce6 micelles in living cells time-lapse sequences TLSM imaging. TLSM images of HeLa cells incubated with HCP@HPE3-Ce6 for 4 h under irradiation for 10 s, 200 s, 5 min, 10 min and 20 min, respectively. Excitation was at 800 nm and the scale bars represent 10 μm. (b) In vitro photocytotoxicity of free Ce6 and HCP@HPE3-Ce6 under 650 nm and 800 nm laser irradiation at 37 o

C and 40 oC, respectively. (c) 1O2 luminescence spectra obtained from oxygen-saturated


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solutions of HCP@HPE3-Ce6 in benzene-d6 under 800 nm at 40 oC; HCP@HPE3-Ce6 in benzene-d6 under 800 nm at 37 oC; HCP@HPE3-Ce6 in benzene-d6 under 650 nm at 37 oC; free Ce6 in benzene-d6 under 800 nm at 37 oC and free Ce6 in benzene-d6 under 650 nm at 37 oC. (d) Decay of optical absorption of ADPA at 378 nm, respectively caused by the generation of 1O2 from HCP@HPE3-Ce6 under 800 nm at 40 oC; HCP@HPE3-Ce6 under 800 nm at 37 oC; HCP@HPE3-Ce6 under 650 nm at 37 oC; free Ce6 under 800 nm at 37 oC and free Ce6 under 650 nm at 37 oC, as a function of laser irradiation exposure time. (e) The cytotoxicity from different laser at 650 nm and 800 nm. (f) The irradiation time-dependent photo-cytotoxicity of HCP@HPE3 micelles, HCP@HPE3-Ce6 micelles and free Ce6 at a Ce6 concentration of 20 μg/mL under the irradiation with 800 nm laser. (g) Caspase-3 protein activity in HeLa cells treated with free Ce6 and HCP@HPE3-Ce6 micelles at a Ce6 concentration of 20 μg/mL for 4 h through 650 nm and 800 nm laser irradiation. All data are presented as the average ± standard error (n = 3).


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Figure 4. In vivo pharmacokinetics and biodistribution of HCP@HPE3-Ce6 micelles. (a) Representative serum concentration-time profiles of free Ce6 and HCP@HPE3-Ce6 micelles (0.10 mmol Ce6 kg-1) after intravenous injection into SD rats (about 200 g) rats. (b) In vivo noninvasive Ce6-NIR fluorescence images of time-dependent whole body imaging of HeLa tumor-bearing nude mice after intravenous injection of free Ce6 and HCP@HPE 3-Ce6 micelles. (c) Ex vivo Ce6-NIR fluorescence images of mice tissues after intravenous injection of free Ce6 and HCP@HPE3-Ce6 micelles (from left to right: heart, liver, spleen, lung, kidney, and tumor). (d) Tissue distribution of Ce6 in HeLa tumor-bearing nude mice after intravenous injection of free Ce6 and HCP@HPE3-Ce6 micelles. All data are presented as the average ± standard error (n = 5).


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Figure 5. In vivo PDT anticancer activity of HCP@HPE3-Ce6 micelles. (a) Schematic illustration of 2P-FRET strategy from HCP@HPE-Ce6 micelles for PDT in tumor areas. (b)


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Body weight changes of HeLa tumor-bearing nude mice after treatment with PBS (non-laser, 650 nm laser, or 800 nm laser), free Ce6 (non-laser, 650 nm laser, or 800 nm laser), and HCP@HPECe6 micelles (non-laser, 650 nm laser, or 800 nm laser). (c) Changes of tumor volume after intravenous injection of PBS (non-laser, 650 nm laser, or 800 nm laser), free Ce6 (non-laser, 650 nm laser, or 800 nm laser), and HCP@HPE-Ce6 micelles (non-laser, 650 nm laser, or 800 nm laser) in HeLa tumor-bearing nude mice. (d) Normalized weight of tumors separated from nude mice after different treatments. (e) The tumor inhibitory rate (TIR) after treatment with PBS (non-laser, 650 nm laser, or 800 nm laser), free Ce6 (non-laser, 650 nm laser, or 800 nm laser), and HCP@HPE-Ce6 micelles (non-laser, 650 nm laser, or 800 nm laser) in HeLa tumor-bearing nude mice. The TIR is calculated using the following equation: TIR (%) = 100 × (mean tumor weight of control group - mean tumor weight of experimental group)/mean tumor weight of control group. Here the control group is PBS with non-laser treatment. The mouse graphic comes from the animals template in ChemBioDraw Ultra 12.0. Data are represented as average ± standard error (n = 5).


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Figure 6. In vivo studies illustrating 2P-PDT therapeutic benefits derived from intravenous injection. Histologic analyses of harvest tumor sections after various treatment were made with: (a) hematoxylin and eosin (H&E, first row, magnification ×200). (b) TUNEL (second row, positive: green, blue color is DAPI staining, magnification ×400). (c) Caspase-3 immunohistogram (third row, positive: brown, magnification ×400). (d) Representative tumor photographs (n = 5 for each group) acquired 20 days after various treatments were shown for quantitative analysis of tumor growth (scale bar: 5 mm).


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TABLE OF CONTENTS (TOC)

(width 9.0 cm × height 4.0 cm)


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Combining Two-Photon-Activated Fluorescence Resonance Energy

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