Redox-Activated Light-Up Nanomicelle for Precise Imaging-Guided

Nov 11, 2016 - College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China. § State Key Laboratory of Indu...
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Redox-Activated Light-Up Nanomicelle for Precise Imaging-Guided Cancer Therapy and Real-Time Pharmacokinetic Monitoring Xingang Liu,†,# Min Wu,†,# Qinglian Hu,‡ Hongzhen Bai,§ Shuoqing Zhang,† Youqing Shen,∥ Guping Tang,*,† and Yuan Ping*,⊥ †

Department of Chemistry, Zhejiang University, Hangzhou 310028, China College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China § State Key Laboratory of Industrial Control Technology, College of Control Science and Engineering, Zhejiang University, Hangzhou 310028, China ∥ Center for Bionanoengineering and State Key Laboratory for Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China ⊥ School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798 ‡

S Supporting Information *

ABSTRACT: Simultaneous tumor imaging, therapy, and pharmacokinetic monitoring can offer a safe and effective strategy for cancer therapy. This work describes the design of a fluorescence light-up nanomicelle that can afford precise imaging-guided drug delivery and pharmacokinetic monitoring in a real-time fashion for cancer chemotherapy. The nanomicelle, which contains a boron dipyrromethene based fluorescent probe as the hydrophobic core and a redox-triggered detachable poly(ethylene glycol) (PEG) shell, can accumulate at the tumor site via enhanced permeation and retention effect. The PEG detachment induced by tumoral and intracellular glutathione can destabilize the nanomicelle, leading to fluorescence light up and simultaneous drug release. Importantly, the fluorescence intensities generated by the nanomicelles in different organs are well-correlated with released drug concentrations in both temporal and spatial manners, suggesting its precise role for imaging-guided drug delivery and pharmacokinetic monitoring in vivo. The tumor growth can be effectively inhibited by the docetaxelloaded nanomicelle formulation, and the nanomicelles are monitored to be excreted via hepatobiliary routes. This nanomicelle for precise imaging-guided chemotherapy provides a safe and robust theranostic strategy for the evaluation of cancer nanomedicine. KEYWORDS: theranostic nanomaterials, drug delivery, fluorescence imaging, reduction-sensitive polymer, BODIPY imaging and the ability to delivery drugs to cancer cells,3 these fluorescently bright nanomaterials commonly suffer from a high nonspecific background and a low signal-to-background ratio (SBR).4 In the past few years, the design of activatible theranostic nanomaterials with the fluorescence “turn on” feature offers opportunities to increase the SBR.5 Ideally, the theranostic probes should be switched from “OFF” to “ON” at the specific site in order to selectively image the target molecules or events. For imaging-guided chemotherapy, these activatable theranostic systems should further be able to efficiently target the tumor through active or passive targeting

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heranostic nanomaterials that can afford simultaneous drug delivery and molecular imaging hold great promise for various diagnostic and therapeutic applications in the dawning era of personalized nanomedicine.1 As both imaging probes and drug delivery vehicles, the theranostic nanomaterials are emerging as an important class of tools which are able to visualize and quantify the pharmacokinetic processes of cellular and molecular therapies, including adsorption, distribution, metabolism, and excretion, at both tissue and cellular levels in a real-time fashion.2 Particularly, imaging-guided drug delivery can trace the pathway of drugs inside the body, evaluate the efficiency of tumor-targeted delivery, and further provide the information regarding the pharmacokinetics of the therapeutics. Whereas a variety of inorganic and organic nanomaterials show intrinsic © 2016 American Chemical Society

Received: October 4, 2016 Accepted: November 11, 2016 Published: November 11, 2016 11385

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Scheme 1. Schematic Illustration of the Behavior and the Fluorescence Recovery Process of BCPSP/DTX Nanomicelles in Vivo and the Chemical Structure of BCPSP

Scheme 2. Synthesis of Target Compound BCPSP

drug delivery capacity have been developed for theranostic purposes.9,10 Most recently, BODIPY-based, bifunctional platinated nanoparticles have been developed for photoinduced tumor ablation.11 Despite the promises of these theranostic systems, the reported imaging capacity and drug delivery procedures are often implemented separately, leading to the low theranostic benefits. In addition, little information about pharmacokinetic processes during the whole therapy is offered, thus failing to obtain the timely safety feedback from the theranostic system. In view of these challenges, we herein develop a redoxactivated light-up nanomicelle for anticancer drug delivery and targeted imaging as well as real-time pharmacokinetic monitoring (Scheme 1). This theranostic system is essentially composed of a redox-triggered, poly(ethylene glycol) (PEG)sheddable amphiphilic polymer, which exhibits a well-defined core−corona structure at its critical micelle concentration (CMC). Whereas a BODIPY fluorophore conjugated with two long alkyl chains is used as the core material, two hydrophilic polymers, PEG and polyethylenimine (PEI) that are linked by redox-responsive disulfide bonds, act as the corona materials

mechanisms, such as enhanced permeation and retention (EPR) effect, and precisely deliver and release the anticancer drugs into cancer cells. To meet these criteria, the nanoparticles should be stable in blood and bear the appropriate size distribution for the EPR process.6 Furthermore, the probe should selectively light up in the tumor tissue or upon the internalization by tumor cells and release the cargo simultaneously to minimize the side effect. Last but not least, the theranostic nanoparticles should be monitored in a realtime fashion to understand the pharmacokinetic process of both drugs and their carriers. Among various small molecular probes, boron dipyrromethene (BODIPY) has been used for various imaging applications due to its excellent photostability, high fluorescence quantum yield, and sharp absorption and emission spectra.7 By taking advantage of its strong hydrophobic properties, BODIPY-based probes have been commercialized as BODIPYFL dyes for staining lipids, membranes, and other lipophilic compounds. In order to improve the water solubility of BODIPY for wider biomedical applications, either amphiphilic BODIPY-based probes alone8 or integrated with 11386

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ACS Nano (Scheme 2). Once the unimer, termed as BCPSP, aggregate and assemble into nanomicelles in a “core−shell” structure at the CMC, the fluorescence of BODIPY is quenched by the driving forces of intermolecular π−π stacking and hydrophobic interaction. As a result, the fluorescence is “OFF” during the systemic ciruclulation. Owing to the nonfouling nature of PEG, the nanomicelles can avoid the nonspecific interaction with serum components, which further allows these nanoparticles for long systemic circulation and to accumulate at the tumor tissue by the EPR effect. However, in the tumoral and intracellular environment,12 the cleavage of disulfide bonds begins on the exofacial surface of tumor cells13 and occurs quickly once upon the nanomicelles are internalized by cancer cells into glutathione (GSH)-elevated milieu. The cleavage of disulfide bonds will not only detach PEG from the micelle surface to promote charge-mediated endocytosis but also accelerate the release of loaded docetaxel (DTX) and expose BODIPY-based fluorophores to the surrounding proteins. The disaggregation of hydrophobic BODIPY moieties can induce a conformational change and increase the molecular rigidity of BODIPY due to the subsequent BODIPY−protein hydrophobic interaction,14 leading to fluorescence “turn on”. As a result, the redoxtriggered light-up nanomicelles not only allow for the imaging of drug accumulation in tumor but also offer the opportunity to monitor the pharmacokinetic process of the nanomicellar system and estimate the drug release and accumulation in different organs, including tumors.

Figure 1. (a) Bathochromic shift of normalized fluorescent spectra of compound 1 (black line) and compound 2 (orange line) in tetrahydrofuran solution. (b) Size distribution of BCPSP/DTX nanomicelles in water determined by dynamic light scattering (the inset is the transmission electron microscopy image). (c) Gel penetration chromatogram (GPC) of BCPSP nanomicelle treated with GSH (10 mM) for 12 h (pink line). Degradation of BCPSP nanomicelle in the presence of GSH was analyzed by GPC. The mobile phase is dimethylformamide with a flow rate of 1.0 mL/min at 60 °C. (d) Critical micelle concentration of the amphiphilic BCPSP determined using pyrene as the fluorescent probe. (e) Fluorescent emission spectra of BCPSP (10 μg/mL) in various organic solvents with different polarity; excitation wavelength = 640 nm. (f) Fluorescent spectra of BCPSP (10 μg/mL) in DMSO/ H2O mixtures with different fractions of H2O (v/v). Excitation wavelength = 640 nm.

RESULTS AND DISCUSSION To construct the BODIPY-based hydrophobic core, we first synthesized intermediate BODIPY dye compound 2 with carboxylic acid ester groups from starting compound 1. As indicated in Scheme 2, compound 2 was synthesized by a Knoevenagel-type condensation reaction between compound 1 and 4-octyloxybenzaldehyde.15 The intermediate compound 2 was further hydrolyzed and reacted with PEI through an amidation reaction to afford compound 3, which was subsequently reacted with disulfide-bearing PEG by N,N′carbonyldiimidazole (CDI) to obtain the target polymer (termed BCPSP). All the synthesized compounds were characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy to confirm their structures (Figures S1− S6). In the 1H NMR spectra of BCPSP (Figure S5), the typical proton signals (around 3.64 ppm) suggest the successful introduction of PEG chains to compound 3. According to the integration of correlated peaks, at least one PEG chain is grafted to compound 3. The multiple peaks from 6.51 to 8.03 ppm are assigned to the BODIPY fluorophore. In the 13C NMR spectrum of BCPSP, the peak around 70.6 ppm also confirms the presence of the repeating unit of PEG. Moreover, the Raman spectrum of disulfide-bearing PEG and native PEG suggests the successful introduction of disulfide bonds onto the distal end of PEG (Figure S7). Concurrently, a non-redoxresponsive analogue, termed BCPP, was synthesized (Figure S8). Interestingly, the fluorescent spectrum of compound 2 shows a bathochromic shift of 150 nm to the near-infrared region, with maximum fluorescence intensity at the wavelength of 670 nm (Figure 1a). Due to the distyryl substitution, compound 2 exhibits an extended π-conjugation system compared to compound 1, thereby leading to the bathochromic shift.16,17 The conjunction of PEI and PEG, however, does not affect the bathochromic shift. Given that the near-infrared region (650−1000 nm) is characterized by minimal tissue

autofluorescence and deep tissue penetration for bioimaging application,16 BCPSP is expected to be more efficient and sensitive over native BODIPY for in vivo imaging. The morphology of the BCPSP was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) (Figure S9). The TEM micrograph and DLS data indicate that BCPSP is able to self-assemble into spherical nanomicelles that mainly distribute from 90 to 130 nm (average size of 101 nm) with a well-defined core−shell structure due to its amphiphilic nature. After the loading of DTX, the average size of nanomicelles slightly increased to 140 nm (Figure 1b). Nevertheless, after the incubation of DTX-loaded BCPSP nanomicelles in 10 mM of GSH for 12 h, the initial spherical micellar structure collapsed, as observed in TEM images (Figure S10). Gel penetration chromatography (GPC) study showed that the elution curve of BCPSP presents a unimodal peak (Figure 1c). However, after the BCPSP polymer was treated with GSH, a strong shoulder peak appeared at the identical elution volume as PEG, which provides strong evidence of the cleavage of disulfide bonds. The CMC of BCPSP nanomicelles was determined to be 6.8 × 10−7 mg/mL (Figure 1d), suggesting the good stability of BCPSP to confer extreme dilution by body fluids.18 As shown in Figure 1e, the 11387

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Figure 2. (a) DTX release from BCPSP nanomicelles with GSH with 10 μM (pink) or 10 mM (yellow) GSH in PBS (pH 7.4), without GSH in PBS (pH 7.4) (black) or acetate buffer solution (pH 5.0) (gray) at 37 °C. The error bars in the graph represent the standard deviations (n = 3). (b) Fluorescent emission spectra of BCPSP nanomicelles (10 μg/mL) in 5% BSA solution (w/w; BSA/PBS) treated with (blue line) or without (gray line) 10 mM GSH or in PBS with 10 mM GSH (yellow line) or the control group BCPP in 5% BSA solution with 10 mM GSH (maroon line); excitation wavelength = 640 nm. (c) Schematic illustration of the mechanism for the fluorescence recovery of BCPSP nanomicelles. (d) HOMO and LUMO distributions of the designated structure from BCPSP, which was calculated with two extreme dihedral angles between meso-aryl substitution and BODIPY fluorophore: 90° (left) and 10° (right).

fluorescence spectra of BCPSP nanomicelles were highly emissive in THF with an emission maximum at 657 nm; however, its fluorescence was dramatically quenched in water solution. In the DMSO/water mixed solvent, the emission intensity of BCPSP declined rapidly with the increase of water fraction from 0 to 30% (v/v) (Figure 1f). When the water fraction is greater than 30%, BCPSP nanomicelles become faintly fluorescent. As water possesses higher polarity over DMSO, the increase of water content leads to the higher polarity of the solvent system and results in aggregation-caused quenching (ACQ) of BODIPY, strongly suggesting the compact and aggregated feature of nanomicellar structure in water. We then investigated the drug release kinetics of DTX from BCPSP nanomicelles. To this end, two concentrations of GSH buffers (10 μM and 10 mM) were used to mimic the extracellular and intracellular GSH level, respectively (Figure 2a). Strikingly, DTX release from BCPSP is through both a diffusion-controlled process and nanomicelle degradation. In the presence of 10 mM GSH, DTX is rapidly released from BCPSP nanomicelles over the first 4 h, followed by sustained release in the following 20 h. Due to the cleavage of disulfide bonds, the BCPSP nanomicelles become unstable and release DTX as a result of BCPSP degradation. However, in the 10 μM GSH, the release is mainly governed by a diffusion-controlled process. The rate of DTX release in the absence of GSH was generally slow and reached a total release of 26 and 37% at pH 7.4 and 5.0, respectively. These results suggest that intracellular

GSH may significantly trigger the rapid release of DTX from BCPSP nanomicelles. Recent studies have shown that BODIPY-based fluorescent probes could serve as a light-up probe for selective sensing of the hydrophobicity of proteins.14 This is based on the mechanism that the hydrophobic interaction between BODIPY-based probes and hydrophobic moieties of a protein will induce the conformational change and the increased molecular rigidity of molecules, which could lead to the fluorescence enhancement. In the current study, we speculate the released BODIPY fluorophores may have the opportunity to contact either extracellular or intracellular proteins to turn on the fluorescence. To test our hypothesis, we studied the fluorescence recovery of BCPSP nanomicelles in the presence or absence of GSH and bovine serum albumin (BSA). BSA was used as the hydrophobic protein model due to its high surface hydrophobicity.14 As shown in Figure 2b, BCPSP nanomicelles were almost nonfluorescent, regardless of before or after the treatment with 10 mM GSH. However, when BCPSP nanomicelles were dissolved in 5% BSA, the fluorescence significantly increased almost 40-fold after the treatment with the same GSH concentration, which is in sharp contrast with those nanomicelles in BSA solution without GSH treatment. In light of these findings, we proposed the possible mechanism for the fluorescence recovery. The process of fluorescence recovery occurs in a stepwise manner: First, after the disulfide bond was cleaved by GSH, the BCPSP nanomicelle degraded and the BODIPY fluorophore in the ACQ state disaggregates. 11388

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Figure 3. (a) Cytotoxicity of blank BCPSP nanomicelle against HeLa cells. The cytotoxicity was evaluated by MTT assay, and PEI (25 kDa) was used as a positive control. Data represent mean ± SD (n = 4, Student’s t-test, ***P < 0.001). (b) Blood hemolytic effect of BCPSP nanomicelles. Red blood cells were treated with BCPSP nanomicelles at various concentrations from 5 to 100 μg/mL. The deionized water was used as a positive control, and 1× PBS buffer solution was used as a negative control. Inset: RBCs treated with BCPSP nanomicelle of different concentration, compared to the water and PBS groups. (c) Binding isotherms of HSA to BCPSP nanomicelle solution determined by isothermal titration calorimetry.

whereas the RBCs remained integral after the treatment with BCPSP nanomicelles (Figure S12). These results suggest that BCPSP nanomicelles are generally hemocompatible. Protein adsorption is one of the key factors to determine the fate of nanoparticles. The strong adsorption of protein over drug carriers will cause opsonization, leading to the subsequent phagocytosis by the macrophages from either the reticuloendothelial system in systemic circulation or in tissues such as the liver and lung.24,25 For a micellar drug delivery system, serum protein adsorption could potentially cause the premature release of drugs from nanomicelles owing to the partition effect before the nanomicelles reach their target sites.26 To study the protein adsorption of BCPSP nanomicelles, BSA was chosen as the model protein,27 and the binding interaction between BSA and the BCPSP nanomicelles was investigated by isothermal titration calorimetry (ITC). The binding constant (K) of BCPSP nanomicelles to BSA is 292 M−1 (Figure 3c), indicating a relative weak serum albumin binding ability.28 Compared with the binding constant of PEI with BSA (1.82 × 104 M−1), the binding constant between BCPSP and BSA is much lower (Figure S13). When the BCPSP unimer forms nanomicelles, the densely packed hydrophilic PEG corona can readily prevent BODIPY from interacting with the hydrophobic pocket of BSA. In such a case, the chance of interaction between BODIPY and BSA is very low. The above results demonstrate that the low fouling nature of BCPSP nanomicelles reduces the nonspecific interactions with serum proteins such as BSA through its steric repulsion effects.23,29 To study the fluorescence recovery after endocytosis, we incubated the BCPSP nanomicelles with HeLa cells for 0.5 h, removed the culture medium, and replenished with the fresh one afterward to remove the excess nanomicelles. The fluorescence recovery was observed after 2 h and became more obvious after 2.5 h (Figure 4a). In order to trace the cellular uptake behavior of BCPSP nanomicelles, HeLa cells incubated with BCPSP nanomicelles were monitored by confocal laser scanning microscopy (CLSM). Cellular uptake study showed a time-dependent increase in fluorescence intensity surrounding the nuclei (Figures 4b and S14), suggesting the efficient internalization capacity of BCPSP/ DTX nanomicelles by tumor cells. Compared with the nonredox-responsive BCPP, the fluorescence intensity was much weaker after the BCPP/DTX nanomicelles were incubated with the cells for 6 h. This suggests that the quenched fluorescence of BCPSP/DTX could only be activated by GSH-induced nanomicelle degradation. As BCPPs are not degraded by GSH, they are still relatively stable at intracellular milieu and have

Subsequently, the disaggregated BODIPY residues can interact with the hydrophobic BSA pocket to form dye−protein complexes (Figure 2c). The hydrophobic dye−protein interaction significantly decreases the polarity of the dye’s microenvironment and reduces the rotations around single bonds in the meso position.19−21 We also calculate the quantum mechanics for the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) gaps through first-principle density functional theory (Figure 2d, Figure S11, and Tables S1−S3). Based on our calculation, we speculate that the degradation of BCPSP nanomicelles disaggregate the BODIPYbased fluorophore, leading to the free rotation of the meso-aryl position of BODIPY. However, once the BODIPY fluorophore interacts with the hydrophobic moiety of a protein, the HOMO−LUMO band gap becomes narrow due to the decrease of nonradiative decay.22 Moreover, the hydrophobic pocket of the protein also reduces the microenvironment polarity of the BODIPY, which also contributes to the fluorescence recovery of the fluorophore.19 Collectively, the decreased HOMO−LUMO band gap, the increased molecular rigidity, and the reduced polarity in the microenvironment of the fluorophore can lead to the remarkable fluorescence enhancement. In order to evaluate its biocompatibility for in vivo application, we first examined the cytotoxicity and blood compatibility of blank BCPSP nanomicelles. The MTT results indicate that blank BCPSP nanomicelles merely bear any detectable cytotoxicity on HeLa cells with the concentration up to 100 μg/mL after 48 h incubation (Figure 3a). After the BCPSP nanomicelles were loaded with DTX, dose-dependent cytotoxicity was observed. At the DTX concentration of 5 μg/ mL, the cell viability was only around 20%, which was lower compared with the equivalent dose of DTX (38%). Blood compatibility is also a critical indicator to evaluate whether the proposed nanomaterials are safe for the in vivo application.23 To study the blood compatibility of BCPSP nanomicelles, we measured the hemolytic potential of BCPSP nanomicelles. The hemolysis rate represents the percentage of the destroyed membrane of red blood cells (RBCs) induced by BCPSP. With a concentration up to 100 μg/mL, BCPSP nanomicelles only induced a hemolysis rate of 3%, indicating its good hemocompatibility.23 We also observed that RBCs treated with water showed red color and other BCPSP solutions up to 100 μg/mL were still clear in the supernatant, which was similar to the PBS control (Figure 3b). Under the microscope, the rupture of RBCs induced by water was clearly observed, 11389

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The DTX-loaded BCPSP nanomicelles also exhibited good ability to induce the apoptosis of cancer cells (Figure 5a,b). In comparison with the apoptosis rate induced by DTX (15.8%), DTX delivered by BCPSP nanomicelles exerted a better proapoptosis effect, leading to an apoptosis rate of 21.7%. At the higher DTX dose (0.500 μg/mL), the apoptosis induced by BCPSP/DTX nanomicelles (46.4%) was even much stronger than that induced by DTX (27.3%). BCPSP/DTX nanomicelles showed a dose-dependent cytotoxicity and exhibited stronger cytotoxicity to HeLa cells with a much lower IC50 (0.172 μg/mL) over DTX (0.642 μg/mL) at the equivalent DTX concentration (Figure 5c). The enhanced cytotoxicity and apoptosis induced by BCPSP/DTX nanomicelles were mainly due to the increased cellular uptake and the rapid disassembly of nanomicelles in response to intracellular GSH environments. To evaluate the effectiveness of drug delivery to tumors, BCPSP/DTX nanomicelles were administered into tumorbearing nude mice via tail vein injections, and the in vivo biodistribution of nanomicelles was first evaluated by tracking the fluorescence of BCPSP. As depicted from the real-time imaging in the living tumor-bearing mice (Figure 6a), most of the nanomicelles accumulated in the liver at 6 h postinjection and then underwent a relative rapid clearance from the liver at 24 h postinjection. However, only faint fluorescence in tumors could be observed at 6 h postinjection. As time further progressed, the fluorescence intensity in the tumor gradually enhanced, reaching the maximum intensity at 12 h postinjection and maintained relatively high fluorescence intensity for a long period. The fluorescence intensity at the tumor site could be distinguished from other surrounding tissues and provided the highest contrast for tumor imaging, demonstrating the tumor targeting through the EPR effect and long retention of BCPSP nanomicelles in the tumor tissue. The low autofluorescence from the tissue background in real-time imaging should be attributed to the near-infrared fluorescence emission of BCPSP. The introduction of vinylbenzene into the BODIPY fluorophore led to the red-shifted emission,31 which could reduce the influence of autofluorescence in tissues.32,33 In order to quantify the BCPSP concentration in vivo, the fluorescence intensity generated by BCPSP was evaluated. Tumors and major organs were further excised at certain times for ex vivo imaging. As illustrated in Figure 6b,c, most of BCPSP/DTX nanomicelles accumulated in the liver and lungs at 6 h postinjection and mainly distributed in the intestine and tumor at 24 h postinjection. This observation clearly indicates that BCPSP/DTX nanomicelles were largely trapped by the mononuclear phagocytic system initially, accumulated at the tumor through EPR effect afterward, and excreted via the hepatobiliary route finally. It should be noted that the mononuclear phagocytic system will compete with the tumor for circulating nanomicelles after systemic administration, and only, on average, 0.7% of the total nanoparticles can finally reach the tumor.34 Ex vivo imaging of intestine tissues excised at various time points, which reflects the process of excretion, has further demonstrated the rapid hepatobiliary excretion route of BCPSP/DTX nanomicelles. As shown in Figure 6d, the fluorescence in the intestine gradually moved toward the anus, and the fluorescent intensity from the intestine decreased with time, becoming almost negligible at 60 h postinjection. These results proved that BCPSP/DTX nanomicelles could be almost completely cleared from the body in 60 h postinjection, which could potentially reduce the side effects caused by unwanted accumulation of therapeutic nanoparticles inside the

Figure 4. (a) Fluorescence images of HeLa cells treated with BCPSP/DTX nanomicelles (1 μg/mL) for 0.5 h. Afterward, the culture medium was removed, and the cells were replenished with the fresh medium to continue the culture for another 2.5 h. (b) Fluorescence images of HeLa cells incubated with BCPSP/DTX nanomicelles for different time length. (c) Three-dimensional images of HeLa multicellular spheroids after incubation with BCPSP/DTX nanomicelles for 6 h. The representative confocal images were taken every 1.25 μm section from the top to the middle of the spheroid. Optical images were taken by CLSM −30, −70, and −130 μm from the spheroid rim. The excitation wavelengths of DAPI and BCPSP are 405 and 633 nm, respectively.

much lower chance of interacting with proteins. In addition, the charge-mediated cellular uptake of BCPP nanomicelles is potentially low as a result of low surface charge. The limited penetration of nanoparticles in tumor tissue constitutes one of the main hurdles for effective cancer therapy. To evaluate the penetration behavior of BCPSP/DTX nanomicelles, HeLa cells were used to culture three-dimensional multicellular spheroids (MCSs) to mimic the morphology and microenvironment of solid tumors.30 The images were taken layer-by-layer to monitor the penetration activities of BCPSP/DTX nanomicelles in MCSs by CLSM. As shown in Figure 4c, the fluorescence signal was observed to primarily distribute on the periphery of the MCSs at the scanning depth of 140 μm, with the increased fluorescence intensity at the scanning depth of 70 μm. However, at the scanning depth of 30 μm, the fluorescence intensity in the interior area of the MCSs increased considerably, implying the good ability of BCPSP nanomicelles to penetrate into deep tumors. The above results were also supported by the reconstructed three-dimensional images of MCSs, where the strong accumulation and retention of BCPSP/DTX inside the MCSs could be clearly observed (Figure S15). 11390

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Figure 5. (a) Cytotoxicity of BCPSP/DTX nanomicelles and free DTX against HeLa cells by MTT assay. Data represent mean ± SD (n = 4, Student’s t-test, *P < 0.05). (b) Apoptosis analysis of HeLa cells after the treatment with the indicated formulations for 24 h. The quantitative analysis was evaluated by means of flow cytometry. Low dose refers to the DTX concentration of 0.1 μg/mL, whereas high dose refers to the DTX concentration of 0.5 μg/mL. Data represent mean ± SD (n = 4, Student’s t-test, **P < 0.01). (c) Relative apoptosis rate of HeLa cells after treatment with different DTX formulations for 24 h.

body. Thus, the current theranostic nanomicellar system could truly reflect the distribution of nanoparticles in vivo with low background. Because the fluorescence recovery is concomitant with drug release, we are interested in exploring whether there is a direct correlation of DTX release with fluorescence intensity. To this end, DTX and BCPSP nanomicelles extracted from different organs including heart, liver, spleen, lung, kidney, and tumor were quantified by high-performance liquid chromatography (HPLC) and fluorospectrophotometry, respectively. After 4 h postinjection, about 30 and 14% of the injected DTX were found to accumulate in liver and kidney (Figure 6e). However, accumulation in the liver and kidneys decreased over time until 6 and 4% at 48 h postinjection. During this period, the amount of DTX in the tumor increased as time progressed, reaching the highest level of 9% at 12 h postinjection. These results are consistent with the biodistribution profile of BCPSP nanomicelles in Figure 6f, where the time-dependent drug distribution in a specific organ seems to be well correlated with its tissue fluorescence. For example, whereas both DTX concentration and fluorescence in the liver gradually decreased with the time, the significant increase was observed at the tumor tissue from 12 to 24 h postinjection, which also indicates that BCPSP/DTX nanomicelles can improve the accumulation of cargo at tumor sites via the EPR effect. Based on the fluorescence intensity and the DTX concentration distributed

in different organs, we established the heat map to reveal the detailed correlation between two variables. As shown in Figure 6g, the spatial and temporal distribution of DTX generally shares the similar response profiles to the fluorescence intensity. To be more specific, the time-dependent DTX distribution in most organs, such as lungs and tumor, exhibits the similar trend to fluorescence intensity generated by BCPSP. The response of fluorescence intensity to DTX concentration in the tumor is positively nonlinear, with the correlation coefficients (r2) of ca. 0.8 (Figure 6h). The response curves in other organs were also established (Figure S16). These data suggest that the spatial and temporal drug accumulation and fluorescence recovery are inter-related, providing a precise approach for monitoring the pharmacokinetic process of the nanomedicine. Given the effective accumulation in tumors, we finally investigated the potential of BCPSP/DTX nanomicelles for cancer treatment in the tumor-bearing nude mice models. As observed in Figure 7a, the tumors from mice treated with saline grow rapidly, whereas the treatment with DTX could moderately inhibit tumor growth. Strikingly, BCPSP/DTX nanomicelles were most effective in inhibiting tumor growth, and the tumor size only reached 273 mm3 on the 21st day of treatment. No body weight loss was observed with the mice treated with BCPSP/DTX nanomicelles during the 21 day treatment, indicating the low systemic toxicity characteristics 11391

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Figure 6. (a) Time-dependent in vivo fluorescence images of BALB/c nude mice with HeLa xenografts. Images were taken at 0, 1, 6, 12, 24, and 48 h after the injection with 200 μL (0.1 mg/mL) of BCPSP/DTX nanomicelles via the tail vein. Then the mice were sacrificed, and the main organs (heart, liver, spleen, lung, kidney, tumor, and intestine) were harvested at different time intervals. Ex vivo imaging of main organs harvested at (b) 6 h or (c) 24 h. (d) Ex vivo imaging of intestine harvested at 12, 24, 36, and 60 h. Quantitative analysis of DTX (e) and fluorescence intensity of BCPSP (f) acquired from tumors and various organs at 4, 6, 12, 24, and 48 h (FL refers to fluorescence intensity, and Con refers to concentration). (g) Heat map of the spatial and temporal changes of the fluorescence intensity of BCPSP and DTX concentration in tumor and other main organs harvested for different time intervals. (h) Response curve between fluorescence intensity of BCPSP and DTX concentration in tumor tissues at different time intervals.

the EPR mechanism, the detachment of PEG triggered by elevated GSH in tumoral milieu may facilitate the tumor penetration and enhance the cellular uptake. The destabilization of nanomicelles, which was mainly triggered by a high level of intracellular GSH, was able to induce the nanomicelle to light up due to the disaggregation of the BODIPY fluorophore and subsequent BODIPY−protein hydrophobic interaction. Although the nanomicelles were primarily distributed in the liver and spleen in the first 4 h after tail vein injection, the BCPSP/DTX nanomicelles could significantly accumulate in the tumor tissue at 12 h postinjection. Because the fluorescence intensity is well correlated with drug concentration in the major organs of tumor-bearing mice, the current system also provides a robust approach for real-time pharmacokinetic monitoring in a temporal and spatial manner. This also directs us to elucidate that the BCPSP/DTX nanomicelles are cleared through hepatobiliary route, thus establishing the pharmacokinetic profile of the nanomicelles for the safe delivery of DTX. The successful inhibition of tumor growth in the mouse model with minimum side effects also proves the effectiveness of BCPSP/ DTX as an anticancer formulation for safe cancer chemotherapy. Collectively, the success of the current study not only defines an effective cancer theranostic strategy but also opens possibilities to integrate the temporal−spatial pharmacokinetic

and minimum side effects of BCPSP/DTX nanomicelle formulation (Figure 7b). The H&E stained sections of tumor tissue from the saline group appeared mostly hypercellular and showed the nuclear polymorphism, whereas the BCPSP/DTX group showed the fewest tumor cells but the highest level of tumor necrosis (Figure 7c). The TUNEL assay also proved that the BCPSP/DTX nanomicelles could induce much more TUNEL-positive cells than the other two formulations. Ki-67 staining assays, which were used to assess tumor proliferation, also showed that the Ki-67 level of BCPSP/DTX treatment group was much lower than that in other groups, implying the lower cell proliferation in tumors but higher antitumor activity induced by BCPSP/DTX nanomicelles. These data confirm that BCPSP can significantly enhance the delivery of DTX to tumors and exert redox-activated drug release to inhibit tumor growth in a more effective manner.

CONCLUSIONS In summary, we developed a BODIPY-based light-up nanomicelle that could offer simultaneous fluorescence imaging and pharmacokinetic monitoring for effective and safe cancer chemotherapy. Whereas PEG could prevent nanomicelles from interacting with serum proteins and facilitate the accumulation of BCPSP nanomicelles in the tumor through 11392

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Figure 7. (a) Tumor growth curve of BALB/c nude mice with HeLa xenografts after four injections of the indicated formulation. Data represent mean ± SD (n = 5, Student’s t-test, *P < 0.05, **P < 0.05). The arrows indicate the injection time. (b) Change of body weight of BALB/c nude mice with HeLa xenografts after the treatment with the indicated formulations. (c) H&E, TUNEL, and Ki-67 analyses of tumor tissues of BALB/c nude mice after the indicated treatment. Dean−Stark apparatus. The solution containing the crude product was concentrated under reduced pressure and purified by silica gel column chromatography (CH2Cl2/hexane = 1:5, v/v) to afford 0.09 g of compound 2 (24% yield): 1H NMR (400 MHz, CDCl3) δ (TMS, ppm) 8.19 (2H, t), 7.58 (6H, m), 7.46 (2H, t), 7.22 (2H, m), 6.92 (4H, t), 6.62 (2H, s), 3.98 (7H, m), 1.81 (4H, m), 1.20−1.50 (26H, m), 0.89 (6H, m); 13C NMR (400 MHz, CDCl3) δ (TMS, ppm) 166.56, 160.14, 153.11, 141.32, 140.28, 136.27, 130.72, 130.24, 129.26, 129.12, 129.03, 117.74, 116.96, 114.83, 77.22, 68.17, 52.38, 31.83, 29.38, 29.25, 26.05, 22.68. Synthesis of Compound 3. Compound 2 (0.79 g, 0.97 mmol) was dissolved in 60 mL of THF/water (1:1, v/v). Then sodium hydroxide (0.40 g, 2.9 mmol) was added, and the mixture was stirred for 24 h at 50 °C. By adding a 10% (v/v) aqueous solution of hydrochloric acid, the mixture was acidified until pH 3−4. The crude mixture was extracted three times with CH2Cl2. The combined organic phases were dried over anhydrous sodium carbonate. The CH2Cl2 was removed by evaporation to afford intermediate compound, which was used without further purification. The intermediate compound (0.08 g, 0.10 mmol), EDCI (0.50 g, 2.6 mmol), and HOAT (0.20 g, 1.4 mmol) were dissolved in 30 mL of anhydrous dichloromethane, followed by addition of 0.2 mL of Et3N. PEI (600 Da, 0.060 g, 0.10 mmol) in 20 mL of anhydrous dichloromethane was added drop-by-drop under nitrogen atmosphere at 0 °C. The reaction mixture was stirred overnight with dialysis in water for 24 h. Then mixture was filtered to remove the precipitate and freeze-dried to yield compound 3 as dark brownish-blue powders (0.06 g, 42% yield): 1H NMR (400 MHz, CDCl3) δ (TMS, ppm) 8.80 (1H, t), 8.42 (1H, t), 7.57 (6H, m), 7.51 (2H, m),7.23 (2H, t), 6.93 (4H, t), 6.62 (2H, s), 4.01 (7H, m), 2.63 (56H, m), 1.81 (4H, m), 1.20−1.50 (26H, m), 0.91 (6H, m); 13C NMR (400 MHz, CDCl3) δ (TMS, ppm) 128.97, 114.29, 77.21, 31.83, 29.70, 29.25, 26.05, 22.67, 14.11. Synthesis of BCPSP. A mixture of PEG-SS (0.021 g, 0.010 mmol) and CDI (0.0030 g, 0.018 mmol) was dissolved in 10 mL of anhydrous

monitoring for more comprehensive evaluation of nanomedicine in the dawning era of personalized medicine.

EXPERIMENTAL SECTION Materials and Instruments. 2,4-Dimethylpyrrole, p-hydroxybenzaldehyde, magnesium perchlorate, piperidine, N-ethyl-N′-(3(dimethylamino)propyl)carbodiimide hydrochloride (EDCI), 1-hydroxy-7-azabenzotriazole (HOAT), 1-chlorooctane, and PEG2000 were obtained from Sigma (St. Louis, MO, USA). Docetaxel (DTX, MW = 807.8792, 99.5%) was purchased from Hao-Xuan Biotechnology Co. Ltd. (Xi’an, China). DAPI was bought from the Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). The starting materials meso-methyl formate-phenyl-BODIPY (compound 1) and PEG-SS were synthesized according to previously reports.15,35 All chemicals were of reagent grade and used without further purification. 1 H NMR and 13C NMR (400 MHz) were recorded on a Bruker DRX-400 spectrometer (Bruker, Ettlingen, Germany). Absorption spectra were measured using a Techcomp UV2310 UV/vis spectrophotometer. Fluorescence measurements were carried out with a Shimadzu RF-6000 spectrofluorophotometer. The average hydrodynamic diameter and ζ-potential were determined at 25 °C by DLS using a 90 Zeta Plus particle size analyzer (Brookhaven Instruments Corp), with a laser light wavelength of 635 nm at a scattering angle of 90°. Particle morphology was observed by TEM (JEM-2010TEM). Briefly, 5 μL of BCPSP nanomicelle solution (0.5 mg/mL) was dropped onto carbon-coated copper grids. After 30 min drying at room temperature, images were recorded by an electron microscope. Synthesis of Compound 2. Briefly, meso-methyl formate-phenylBODIPY (compound 1) (0.19 g, 0.50 mmol) and 4-octyloxybenzaldehyde (0.70 g, 3.0 mmol) were first dissolved in 30 mL of anhydrous toluene, followed by addition of piperidine (0.2 mL), acetic acid (0.2 mL), and a small amount of Mg(ClO4)2. The reaction mixture was stirred and refluxed for 72 h in a round-bottom flask equipped with a 11393

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serum. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. For microscopic observation, 5 × 104/well HeLa cells were seeded onto 24-well plates and incubated at 37 °C for 18 h. After being coincubated with 1 μg/mL of BCPSP nanomicelles in fresh culture medium at 37 °C for 4 h, cells were washed twice with ice-cold PBS and counterstained with DAPI to label nuclei. Coverslips were mounted on slides and analyzed by CLSM (Radiance2100, Bio-Rad). To monitor the fluorescence recovery of BCPSP, culture medium with BCPSP nanomicelles was removed after being co-incubated for 0.5 h and fresh culture medium was added. Then the cells were visualized at interval time with CLSM. Culture medium with BCPSP nanomicelles without removal was set as the control. Cellular Uptake of BCPSP Nanomicelles in Multicellular Spheroids. The three-dimensional MCSs were cultured according to the method previously described with minor modifications.36,37 Briefly, the prewarmed culture dish was covered by 10 mL of hot agarose solution (1.5 w/v %) and then cooled to form a layer of agaropectin. HeLa cells were seeded at a density of 1 million per dish with 12 mL of DMEM medium. Cells were incubated at 37 °C in humidified atmosphere with 5% CO2, and the culture medium was replaced every other day. MCSs formed spontaneously after 5 days. MCSs with a diameter of about 250−350 μm were harvested and handpicked with a Pasteur pipet and transferred to a 5 mL Eppendorf tube. BCPSP/DTX nanomicelles (1 μg/mL) in fresh culture medium were cocultivated with the spheroids for 4 h. The medium was removed, and the spheroids were rinsed with PBS. The spheroids were imaged immediately by CLSM (Radiance2100, Bio-Rad). The representative confocal images were taken every 1.25 μm from the top to the middle of spheroid. In Vitro Cytotoxicity Study. The cytotoxicity of BCPSP nanomicelles was assessed by MTT assay. The HeLa cells were seeded in 96-well plates at a density of 8000 cells/well for 18 h. The culture medium was then replaced by a serum-free medium containing various concentrations of free DTX, BCPSP/DTX nanomicelles, BCPSP nanomicelles, and PEI (25 kDa). After 48 h incubation, the medium was replaced with the MTT solution (0.5 mg/mL in serumfree DMEM medium) for 4 h. After removal of MTT medium, the formazan crystals were dissolved in 100 μL of DMSO and the microplates were agitated for 10 s at a medium rate prior to the spectrophotometrical measurement at a wavelength of 570 nm on an ELISA reader (model 680, Bio-Rad). The untreated cells served as the 100% cell viability control, and the completely died cells served as the blank. The relative cell viability (%) related to control cells was calculated by the formula below:

DMSO, followed by addition of 0.3 mL of Et3N and stirred at room temperature for 4 h. Then compound 3 (0.014 g, 0.010 mmol) in 10 mL of anhydrous DMSO was added dropwise over 3 h with vigorous stirring, followed by reaction for an additional 12 h. After that, the mixture was dialyzed using a dialysis tube (MWCO = 3500) in water for 2 days and freeze-dried to yield the desired product BCPSP (0.023 g, 67% yield) as a black blue powder: 1H NMR (400 MHz, CDCl3) δ (TMS, ppm) 7.81 (2H, t), 7.55 (6H, m), 7.43 (2H, t), 7.22(2H, m), 6.91 (4H, t), 6.61 (2H, s), 3.98 (7H, m), 3.64 (120H, m), 3.37 (3H, s), 2.63 (30H, m), 1.78 (4H, m), 1.20−1.50 (26H, m), 0.89 (6H, m); 13 C NMR (400 MHz, CDCl3) δ (TMS, ppm) 151.74, 129.47, 120.97, 77.20, 70.57, 31.80, 29.71, 26.06, 22.67, 14.13. Preparation of BCPSP Nanomicelles and Evaluation of Critical Micelle Concentration. The BCPSP nanomicelles were prepared via a film dispersion method. Briefly, 5 mg of BCPSP compounds was dissolved in 10 mL of dichloromethane. The solvent was removed by vacuum rotary evaporation to form a dry film. The dried film was then hydrated with 10 mL of PBS buffer (10 mM, pH 7.4) for 10 min under stirring, followed by sonication for 50 min. The solution was filtered through a 0.45 μm filter. Pyrene was used as a fluorescent probe to determine the CMC of BCPSP. A serial BCPSP solution ranging from 1 × 10−9 to 5.0 × 10−6 mol/L was prepared. Then the solutions were added to flasks containing the fluorescent probe pyrene, with the final concentration of pyrene being 6.0 × 10−7 mol/L in water. The solutions were then sonicated for 30 min and kept overnight at room temperature to prepare the nanomicelle formation. The fluorescent spectra were measured at the excitation wavelength of 335 nm on a fluorescence spectrophotometer (Shimadzu RF-6000). Excitation and emission bandwidths were 5 nm. The intensity ratios of I373/I384 from the excitation spectra were plotted against the BCPSP concentration to determine the CMC. Preparation of DTX-Loaded Nanomicelles. A mixture of 5 mg of BCPSP and 1 mg of DTX was dissolved in 10 mL of dichloromethane. The solvent was removed by vacuum rotary evaporation to form a dry film. The dried film was then hydrated with PBS buffer (10 mM, pH 7.4) and stirred for 10 min. The solution was sonicated for 50 min and filtered through a 0.45 μm filter to remove the unloaded DTX and then lyophilized. To determine drug encapsulation efficiency (EE) and drug loading content (LC), lyophilized BCPSP nanomicelle powders were dissolved in acetone and centrifuged. The amount of encapsulated DTX in the supernate was detected by a HPLC system with a reverse-phase C18 column. The mobile phase consisted of acetonitrile−water (70:30, v/ v) at a rate of 0.6 mL/min at 25 °C. An ultraviolet detector was set to 230 nm. LC and EE were then calculated according to the following equations:

V% =

weight of DTX in micelles EE (%) = × 100% weight of DTX in feed

LC (%) =

[A]experimental − [A]blank [A]control − [A]blank

× 100%

where V% is the percent of cell viability, [A]experimental is the absorbance of the wells culturing the treated cells, [A]blank is the absorbance of the blank, and [A]control is the absorbance of the wells culturing untreated cells. Cell Apoptosis Assays. Apoptosis was measured by annexin VFITC/PI double staining according to the apoptotic assay kit. HeLa cells cultured in 6-well plates were treated with blank BCPSP nanomicelles, DTX (0.1 and 0.5 μg/mL), and BCPSP/DTX nanomicelles (equivalent to 0.1 and 0.5 μg/mL DTX) for 24 h. The cells were digested and washed with PBS buffer. The collected cells were resuspended in 500 μL of binding buffer and stained with 5 μL of annexin V-FITC and 5 μL of PI for 15 min in the dark. The stained cells were analyzed by flow cytometry (BD FACS Calibur). HSA Binding with BCPSP Nanomicelles. The interactions of BCPSP nanomicelles and protein were analyzed by isothermal titration calorimetry (MicroCal ITC200 microcalorimeter). Human serum albumin (HSA) was chosen as the model protein. BCPSP nanomicelle solution (1.4 mL, 0.014 mg/mL) was first titrated with successive injections of HSA solution (100 μM) under stirring at a speed of 1000 rpm. The titration volume of the first injection was 5 μL, followed by 20 injections of 10 μL. The spacing between each injection was 90 s.

weight of DTX in micelles × 100% weight of DTX‐loaded micelles

In Vitro Drug Release. The release of DTX from BCPSP/DTX nanomicelles triggered by biological stimuli was studied using a simple dialysis method under different conditions: (i) PBS (10 mM, pH 7.4), (ii) acetate buffer solution (10 mM, pH 5.0), (iii) PBS (10 mM, pH 7.4) containing 10 μM GSH, and (iv) PBS (10 mM, pH 7.4) containing 10 mM GSH. Briefly, 2 mL of BCPSP/DTX nanomicelle solution containing 0.16 mg of DTX was first placed into a dialysis bag (molecular weight cutoff = 1000) and dialyzed against corresponding buffer medium (100 mL) in an incubation shaker at 37 °C at 100 rpm. At selected time intervals, 0.1 mL of release medium was taken out for HPLC analysis (using the same parameters as mentioned above), while the same amount of fresh buffer solution was added back to the original release medium. Cellular Uptake Study. HeLa cells were purchased from American Type Culture Collection (Rockville, MD) and cultured in RPMI DMEM culture medium supplemented with 10% fetal bovine 11394

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ACS Nano The solutions were stirred at 25 °C at a reference power of 10 μcal/s. Heats of dilutions were corrected by subtracting values for nanomicelle-free blank solutions. A one-site binding model was used to fit the binding isotherms. Hemolysis Experiment. The blood compatibilities of BCPSP were evaluated by a hemolysis test on mouse RBCs. The mouse RBCs were separated according to the literature.18 Briefly, the anticoagulated blood was diluted with PBS and centrifuged at 1500 rpm for 10 min at 4 °C. The erythrocyte pellets at the bottom of the centrifuge tube were collected and washed three times with 1× PBS. The resultant RBC suspension was diluted with PBS, producing a stock RBC solution. RBCs upon incubation with water and 1× PBS were used as positive and negative controls, respectively. A total of 0.5 mL of RBC stock solution was incubated with different concentrations of BCPSP, and the volumes of the mixtures were adjusted to 1.0 mL with 1× PBS. The resultant mixtures were incubated at 37 °C for 4 h and centrifuged at 1500 rpm for 10 min. The absorbances of the supernatant solution of test (Atest), positive control (Apos), and negative control (Aneg) at 540 nm were measured using a UV−vis spectrophotometer. Each set of experiments was carried out three times. The hemolysis (%) = [(Atest − Aneg)/(Apos − Aneg)] × 100%. Animals Models. Athymic female mice (BALB/c strain) (4−6 weeks old, 18−22 g) were purchased from the Zhejiang Chinese Medical University and maintained in a pathogen-free environment under controlled humidity and temperature. The animal experiments were performed in accordance with the CAPN (China Animal Protection Law). The xenograft tumor model was generated by subcutaneous injection of 1 × 106 HeLa cells into the abdomen of BALB/C mice. In Vivo Fluorescence Imaging. Roughly 2−3 weeks after tumor model xenograft (once tumors reached −8 mm diameter), imaging experiments were initiated. Two hundred microliters of BCPSP/DTX nanomicelle solution (0.1 mg/mL) was injected into HeLa-tumorbearing mice through the tail vein. In vivo fluorescence imaging was performed at different time points postinjection using a Maestro2 in vivo imaging system (Cambridge Research & Instrumentation, CRi, Woburn, MA). The mice were anesthetized with isoflurane and imaged at the indicated time points. The excitation wavelength was set at 605 nm, and the emission wavelength was chosen from 640 to 820 nm. At 24 h postinjection, the mice were sacrificed. The tumor and normal tissues (heart, lung, kidney, spleen, and liver) were excised and washed with 0.9% NaCl for the ex vivo fluorescence imaging. Pharmacokinetics and Biodistribution Studies. To evaluate the pharmacokinetics, HeLa-tumor-bearing mice were injected with BCPSP/DTX nanomicelles in PBS (0.1 mg/mL) via the tail veins at a final volume of 200 μL. At predetermined time points (4, 6, 12, 24, 48 h), the mice were sacrificed and the blood samples were collected, heparinized, and centrifuged to obtain the plasma. Various organs were excised and weighed. The organs and plasma were suspended in acetonitrile, intensely homogenated, and filtrated. Drug concentration was subsequently quantified by HPLC. The fluorescence intensity of BCPSP was measured at 670 nm with an excitation wavelength of 610 nm. The concentrations of probes or drug in the blood and various organ samples were determined individually according to standard curves. In Vivo Antitumor Efficacy. BALB/c nude mice bearing HeLa tumors were randomly divided into four groups (n = 5) when the tumors grew to around 80−100 mm3. Tumor volumes were estimated using the spherical tumor volume formula V = π/6 × ab2, where a represents length (mm) and b represents width (mm). The mice were treated by intravenous injection of saline, BCPSP, DTX, and BCPSP/ DTX (equivalent to 5 mg/kg of DTX) at 1, 5, 9, and 13 days. The tumor size and body weight of mice were recorded every 4 days for six times. After observation for 21 days, mice were sacrificed and tumor tissues were fixed with 4% paraformaldehyde overnight at 4 °C and embedded in paraffin for analysis. Tissue sections (6 μm) were stained with hematoxylin/eosin (H&E). The terminal transferase dUTP nickend labeling (TUNEL) assay was applied to further detect the cell apoptosis in tumor tissues according to the manufacturer’s instructions (Roche, Basel, Switzerland). The expression levels of Ki-67 in tumor

tissues after different treatments were conducted by immunohistochemical staining. All sections were examined on a Leica (DMLB & DMIL) microscope. Computational Methods. All calculations were carried out with Gaussian 09. To identify the mechanism responsible for the selective enhancement of fluorescence behavior of the BCPSP, we used density functional theory that employs a range separated hybrid functional HSEH1PBE for the exchange and correlation to carry out the optimization and electronic structure calculations. For the all-electron Gaussian basis set, 6-311G(d,p) was used for all calculations. To include the solvent effect due to water or ethanol, we used the polarizable continuum model with default parameters in Gaussian 09.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06688. Synthesis procedures, characterization data for BCPSP, other additional in vitro data, and DFT calculations (PDF)

AUTHOR INFORMATION Corresponding Authors

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

Hongzhen Bai: 0000-0002-0886-3906 Guping Tang: 0000-0003-3256-740X Author Contributions #

X.L. and M.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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