Fabrication of pH-Responsive Nanoparticles with an AIE Feature for

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Fabrication of pH-Responsive Nanoparticles with an AIE Feature for Imaging Intracellular Drug Delivery Xing Wang, Yanyu Yang, Yaping Zhuang, Peiyuan Gao, Fei Yang, Hong Shen, Hongxia Guo, and De-Cheng Wu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00744 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 23, 2016

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Fabrication of pH-Responsive Nanoparticles with an AIE Feature for Imaging Intracellular Drug Delivery Xing Wang,† Yanyu Yang,†,‡ Yaping Zhuang,†,‡ Peiyuan Gao,†,‡ Fei Yang,†,‡ Hong Shen,† Hongxia Guo†,‡ and Decheng Wu*,†,‡ †

Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics

& Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡

University of Chinese Academy of Sciences, Beijing 100049, P.R. China

KEYWORDS: TPE, tadpole-shaped polymer, pH-responsive nanoparticles, cellular imaging, drug delivery.

ABSTRACT: Here we demonstrated a facile method to construction of self-assembled nanoparticles with excellent fluorescent property by synergetic combination of unique AIE effects and tadpole-shaped polymers. The introduction of acid-sensitive Schiff base bonds furnished the polymeric vesicles and micelles unique pH responsiveness that can possess maximal drug-release controllability inside tumor cells upon changes in physical and chemical environments but present good stability under physiological conditions. Benefited from the

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efficient fluorescence resonance energy transfer (FRET), the DOX-loaded fluorescent aggregates were employed for intracellular imaging and self-localization in surveillance of systemic DOX delivery. Cytotoxicity assay of the DOX-loaded aggregates indicated a fast internalization and a high cellular proliferation inhibition to MCF-7 cells while the PEG-POSS-(TPE)7 nanoparticles displayed no cytotoxicity, exhibiting excellent biocompatibility and biological imaging property. These results indicated these biodegradable nanoparticles, as a class of effective pH-responsive and visible nanocarriers, have a potential to improve smart drug delivery and enhance the antitumor efficacy for biomedical applications.

INTRODUCTION Smart nanovehicles such as micelles, vesicles, liposomes and inorganic materials1-3 have been widely employed as the promising drug delivery systems for cancer chemotherapy on account of their improved pharmacokinetics and pharmacodynamics arising from the enhanced permeation and retention (EPR) effect.4,5 Generally, these self-assembled nanoparticles had several significant advantages of suitable dimension, stealthy surface, increased water solubility, long in vivo circulation times, efficient bioavailability and enhanced drug stability in blood circulation.6 Unfortunately, the side effects of the drug carriers mainly included slow drug release and invisible carrier track after they were entered into the cells. So, it is desirable to develop novel delivery systems that can help the drug target tumors, but also realize in real-time monitor of drug localization and release upon internalization into the cancer cells. The research on fluorescent nanoparticles had attracted considerable interest for optoelectronics, biomedical actuators, cell imaging, diagnostic sensors, and targeted drug


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delivery.7,8 In general, the popular methodology was integration of fluorescent dyes into selfassembled skeletons to construct visible drug delivery systems. However, encapsulation of dyes may induce morphological transition of nanostructures, and were also apt to burst release on account of the instability and hydrolysis of the assemblies.9 As an alternative strategy, great efforts have been made towards synthesis of pi-conjugated or dye-labeled amphiphiles to develop fluorescent nanovehicles in recent years. However, most of conventional fluorophores were toxic and their hydrophobic planar structures always induced strong intermolecular π-π interactions, resulting in fluorescent quenching and photobleaching in an aggregated state because of the aggregation-caused quenching (ACQ) effect.10,11 To completely eliminate the ACQ effect, aggregation-induced emission (AIE) luminogens, as an exotic class of luminophores, showed a great promise in biosensing and bioimaging.12-14 They emitted weak luminescence in a molecularly dissolved state but exhibited strong fluorescence in an aggregated state. The main principle of AIE mechanism is due to the restriction of free intramolecular rotation of the phenyl rings and prohibition of energy dissipation via nonradiative channels. Taking advantage of the unique AIE property, development of fluorescent drug delivery systems based on AIE fluorogens is being considered as a promising strategy for simultaneous imaging diagnosis and cell therapy. A pH-responsive nanoparticle was furnished for specific tumor targetability and maximal drug release controllability inside the target cells upon changes in physical and chemical environments.15 On account of the increased fermentative metabolism and poor perfusion, tumor microenvironment has an acidic pH (pH 6.5-7.2) relative to the normal tissue under a physiological condition (pH 7.2-7.4). Once being endocytosized, drug-carriers encounter a gradient pH from early endosomes (pH 6.0-6.5), late endosomes (pH 5.0-6.0) to lysosomes (pH 4.0-4.5). By means of this natural cue, pH-responsive drug delivery systems have been fabricated


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to carry drug molecules at a physiological pH, and accelerate their release at the tumor sites or specific organelles to satisfy the therapeutic drug concentrations within tumor cells.16 Therefore, insertion of acid labile linkages within the polymer stretch is an effective method for building pH-responsive nanoparticles. Herein, a smart pH-responsive drug delivery system was fabricated for cellular imaging and pH-triggered drug release based on the fluorescent nanoparticles by tadpole-shaped PEG-POSS(TPE)7 polymers. By adjusting the length of hydrophilic PEG chains, the amphiphilic PEGPOSS-(TPE)7 self-assembled into vesicles and micelles in aqueous solutions (Scheme 1). TPE groups were employed as hydrophobic AIE-based fluorophores via Schiff base bonds, furnishing the assemblies strong fluorescence in the aggregate state. The bridged Schiff bases can maintain structural integrity at normal physiological condition but render the aggregates disassembly in an acidic condition. Notably, incorporation of rigid cage-shaped POSS units17 can highly restrict TPE intramolecular rotation that powerfully improved AIE effects in the aggregate state. After encapsulation of antitumor DOX into the polymeric micelles or vesicles, these fluorescent aggregates could preserve stability and minimize the payload leakage in blood circulation, but rapidly release the DOX due to the protonation of Schiff base after the internalization by tumors, which could be easily in situ traced during the whole process of drug delivery. Scheme 1. Schematic Illustration of Self-Assembled Vesicles and Micelles.


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EXPERIMENTAL SECTION Materials. Trifluoromethanesulfonic acid (99%, Aldrich), octavinyl POSS (98%, Hybrid Plastics), poly(ethylene glycol) methyl ether (PEG-OH, Mn = 350 g/mol and 1900 g/mol, Alfa Aesor), 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%, J&K), 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride (EDCI, 99%, Energy Chemical), 2-mercaptoethylamine hydrochloride (98%, Energy Chemical), 4-(dimethyl-amino)-pyridine (DMAP, 99%, Aldrich), 4phenylazophenol (98%, Alfa Aesar), 1,3-dibromopropane (99%, J&K), thiocarbamide (99%, J&K), succinic anhydride (99%, J&K), bromotriphenylethylene (99%, Energy Chemical), 4formylphenylboronic acid (99%, Energy Chemical), tetrabutyl ammonium bromide (TBAB, 99%, Energy Chemical), tetrakis (triphenylphosphine) palladium (0) (99%, Energy Chemical), triethylamine (TEA, 99%, Beijing Chemical Works), sodium hydroxide, potassium carbonate, hydrochloric acid, ethanol, ethyl acetate, n-hexane and ether (reagent grade, Beijing Chemical Works), tetrahydrofuran (THF) and dichloromethane (CH2Cl2) were purified by stirring over calcium hydride for 24 h followed by distillation. All other reagents were purchased from SigmaAldrich and used as received without further purification. Characterizations. 1H and 13C NMR spectra were obtained on a Bruker DRX-400 spectrometer in chloroform-d using tetramethylsilane (TMS) as internal reference. Gel permeation


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chromatography (GPC) measurements were carried out on GPCmax VE-2001 (Viscotek) equipped with a Viscotek TriSEC Model 302 triple detector array (refractive index detector, viscometer detector, and laser light scattering detector) using two I-3078 Polar Organic Columns. THF was used as the eluent at a flow rate of 1.0 mL min-1. Molecular weight (Mn and Mw) and polydispersity indexes (PDI) were obtained using the workstation software equipped with the system by the processing method for dendritic polymers based on a working curve of polystyrene standards. Transmission electron microscopy (TEM) images were obtained on a JEM-2200FS microscope (JEOL, Japan). A 5 μL droplet of assembled solution was dropped onto a copper grid (300 mesh) coated with a carbon film, followed by drying at room temperature. Atomic force microscopy (AFM) images were obtained on a Nanoscope Multimode III AFM instrument (Vecco, America) in tapping mode. Dynamic light scattering (DLS) spectra were obtained on a commercial laser light scattering spectrometer (ALV/DLS/SLS-5022F) equipped with a multi-τ digital time correlator (ALV5000) and a cylindrical 22 mW UNIPHASE He-Ne laser (λ0=632.8 nm) was used. All data were averaged over three time measurements. The laser light scattering cell is held in a thermostat index matching vat filled with purified and dust-free toluene, with the temperature controlled to within 0.1 °C. Fluorescence measurements were carried on a Hitachi F4600 photo-luminescent spectrometer with a xenon lamp as a light source. Cellular uptake was imaged by a confocal laser scanning microscopy (CLSM, Leica TCS-SP8) with excitation at 405 nm for TPE and 488 nm for DOX. Synthesis of Septvinyl Monohydroxyl POSS, (vinyl)7-POSS-OH. To a solution of octavinyl POSS (3.8 g, 6 mmol) in 200 mL of fresh distilled CH2Cl2, trifluoromethanesulfonic acid (0.54 mL, 6 mmol) was slowly added via syringe at room temperature. After 4 hours, the solution was diluted with CH2Cl2 and then washed with saturated aqueous Na2CO3 three times. The organic


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layer was then concentrated by evaporating the excess solvent. Then wet acetone was added into the concentrated solution and the hydrolysis process was performed for 6 h. The crude product was yielded after removing the solvent. The final product was separated chromatographically on silica gel with CH2Cl2/n-hexane (v/v = 2/1) to afford the white solid with 32.1% yield. 1H NMR (CDCl3, 400 MHz, ppm): δ 1.62 (s, 1H), 1.22 (t, 2H), 3.82 (t, 2H), 5.85-6.16 (m, 21H). 13C NMR (CDCl3, 400 MHz, ppm): δ 17.3, 58.5, 128.6, 137.2. The preparation of tadpole-shaped polymers based on hydrophiphilic PEG chains with two molecular weights (Mn = 350 g/mol and 1900 g/mol) was very similar, so we selected PEG-OH (Mn = 1900 g/mol) as an example to depict the synthesis methods and characterizations while PEG350-POSS-(TPE)7 was reported in our previous work.18 Synthesis of the Carboxyl-Terminated mPEG, PEG1900-COOH. PEG-OH (Mn = 1900 g/mol, 9.5 g, 5 mmol) and TEA (0.7 mL, 5 mmol) were dissolved in 60 mL of fresh distilled CH2Cl2, and then succinic anhydride (0.8 g, 8 mmol) was added into the solution. After stirring at room temperature for 6 h, the mixture was washed with 2 M HCl aqueous solution, saturated NaCl aqueous solution and DI water for three times and dried over anhydrous MgSO4. After evaporating the solvent, the residue was dissolved in CH2Cl2 and was precipitated in cold ether for three times to afford the white solid with 95.8% yield. 1H NMR (CDCl3, 400 MHz, ppm): δ 2.59-2.63 (m, 4H), 3.38 (s, 3H), 4.21 (t, 2H).

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C NMR (CDCl3, 400 MHz, ppm): δ 29.5, 30.2,

58.5, 63.6, 69.1, 71.6, 72.1, 171.6, 176.8. Synthesis of the Tadpole-Shaped Polymer, PEG1900-POSS-(vinyl)7. PEG1900-COOH (1 g, 0.5 mmol), (vinyl)7-POSS-OH (0.3 g, 0.46 mmol), EDCI (0.11 g, 0.55 mmol) and DMAP (12 mg, 0.1 mmol) were added to a 100 mL round-bottomed flask equipped with a magnetic stirring bar,


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followed by the addition of 50 mL of freshly dried CH2Cl2 to fully dissolve all the solids. The solution was cooled to 0 °C for 30 min and allowed to the room temperature, and then further stirred for another 24 h to complete the reaction. Then, the solution was washed with 2 M HCl aqueous solution, saturated NaCl aqueous solution and DI water for three times and dried over anhydrous MgSO4. The final product was precipitated into ether for several times to remove the excess (vinyl)7-POSS-OH to afford the white powder with 72.1% yield. 1H NMR (CDCl3, 400 MHz, ppm): δ 1.19 (t, 2H), 2.64 (m, 4H), 3.38 (s, 3H), 3.83 (t, 2H), 4.26 (t, 2H), 5.85-6.16 (m, 21H). 13C NMR (CDCl3, 400 MHz, ppm): δ 12.6, 28.4, 28.6, 58.1, 60.3, 63.1, 68.2, 71.2, 128.5, 136.2, 172.9. Synthesis of the Tadpole-Shaped Polymer, PEG1900-POSS-(NH3Cl)7. PEG1900-POSS-(vinyl)7 (0.27 g, 0.1 mmol), 2-mercaptoethylamine hydrochloride (0.12 g, 1 mmol), DMPA (25 mg, 0.1 mmol) were dissolved in 20 mL mixture solution of THF/MeOH (v/v = 1/2). After irradiation under a 365 nm UV lamp at room temperature for 6 h to make sure no vinyl groups existed. The product was precipitated into ether for several times and then purified by ultrafiltration (MWCO 1000) and collected after freeze-drying to afford the white powder with 66.5% yield. 1H NMR (MeOD, 400 MHz, ppm): δ 1.08 (t, 14H), 1.19 (t, 2H), 2.64 (m, 4H), 2.66 (t, 14H), 2.81 (t, 14H), 2.96 (t, 14H), 3.38 (s, 3H), 3.83 (t, 2H), 4.24 (t, 2H), 8.29 (s, 21H). 13C NMR (MeOD, 400 MHz, ppm): δ 12.2, 12.7, 25.2, 28.0, 28.2, 28.6, 39.0, 58.1, 60.3, 63.1, 68.2, 71.1, 173.6. Synthesis of Monomer, TPE-CHO. Bromotriphenylethylene (2.01 g, 6 mmol) and 4formylphenylboronic acid (1.35 g, 9 mmol) were dissolved in the mixture of toluene (40 mL), TBAB (0.19 g, 0.6 mmol) and 1.2 M potassium carbonate aqueous solution (10 mL). The mixture was stirred at room temperature for 0.5 h under Ar gas followed adding Pd(PPh 3)4 (60 mg, 5.3×10-3 mmol) and then heated to 90 °C for 24 h. After that the mixture was poured into


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water and extracted three times with ethyl acetate. The organic layer was dried over anhydrous MgSO4. After evaporation the solvent under reduced pressure, the residue was chromatographed on a silica gel column with CH2Cl2/n-hexane (v/v = 1:2) as an eluent to give TPE-CHO (1.95 g, 90.1% yield). 1H NMR (CDCl3, 400 MHz, ppm): δ 7.02-7.03 (m, 6H), 7.11 (m, 9H), 7.18 (d, 2H), 7.60-7.63 (d, 2H), 9.90 (s, 1H). 13C NMR (CDCl3, 400 MHz, ppm): δ 126.9, 127.1, 127.7, 127.9, 129.2, 131.3, 134.3, 139.8, 143.0, 150.6, 191.9. Synthesis of the Tadpole-Shaped Polymer, PEG1900-POSS-(TPE)7. PEG1900-POSS-(NH3Cl)7 (0.21 g, 0.06 mmol) and TEA (84 μL, 0.6 mmol) were dissolved in 25 mL of ethanol. Then 10 mL of ethanol containing TPE-CHO (0.22 g, 0.6 mmol) was dropwise added into the solution under a nitrogen atmosphere. The reaction mixture was degassed three times with nitrogen and refluxed at 80 °C for 12 h to produce yellow precipitates. The crude products were obtained after filtration, washing with cold MeOH and drying to afford brilliant yellow powder with 61.3% yield. 1H NMR (CDCl3, 400 MHz, ppm): δ 1.14 (t, 14H), 1.22 (t, 2H), 2.70 (m, 18H), 2.90 (t, 14H), 3.38 (s, 3H), 3.78 (t, 14H), 4.27 (t, 2H), 6.89-7.62 (m, 133H), 8.11 (s, 7H).

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C NMR

(CDCl3, 400 MHz, ppm): δ 13.2, 13.5, 26.4, 29.1, 32.8, 58.5, 59.5, 60.8, 70.3, 71.3, 129.6, 127.8, 129.1, 130.8, 134.1, 140.6, 141.6, 143.7, 146.4, 162.2, 172.3. Formation and Self-Assembly of the Amphiphilic Tadpole-Shaped Polymer. A typical selfassembly aggregate solution was prepared as following: PEG-POSS-(TPE)7 (5 mg) was first dissolved in THF (1 mL), which is a good solvent for POSS, TPE and PEG components. Then deionized water (4 mL) was added dropwise into the solution at the rate of 0.05 mL/min via a syringe pump. The colloidal dispersion was further stirred for another 1 h and the temperature was fixed at 25 °C during the self-assembling process. The organic solvent was removed by dialysis (MW cutoff, 1 kDa) against deionized water for 3 days.


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Determination of pH-Triggered Destabilization of PEG-POSS-(TPE)7 Nanoparticles. For determining the pH sensitivity of PEG-POSS-(TPE)7 nanoparticles, a certain amount of phosphate buffered saline (PBS) with different pH values (5.0 and 7.4) was added into 10 mL solution of PEG350-POSS-(TPE)7 and PEG1900-POSS-(TPE)7 nanoparticles (1 mg/mL), and then the solution was mildly stirring at 37 °C. After several minutes, the size changes of nanoparticles were measured by DLS measurement. In addition, a certain amount of PBS with different pH values (pH = 5.0, 5.8, 7.4 and 10.0) was added into 10 mL solution of PEG-POSS-(TPE)7 nanoparticles. After mildly stirring at 37 °C for the appointed time, fluorescent intensities of nanoparticles were measured by fluorescence measurement. Preparation of DOX-Loaded PEG-POSS-(TPE)7 Aggregates. DOX-loaded PEG-POSS(TPE)7 aggregates were prepared as follows: 20 mg PEG-POSS-(TPE)7 was dissolved in 1 mL of DMF, followed by adding a predetermined amount of DOX∙HCl and 1.5 molar equiv of triethylamine and stirred at room temperature for 2 h. Then 4 mL of PBS (pH 7.4) was added dropwise into the solution at the rate of 0.05 mL/min via a syringe pump. Subsequently, the solution was dialyzed against deionized water for 24 h (MW cutoff, 4 kDa) to remove free DOX and byproducts, followed by lyophilization to obtain the freeze dried DOX-loaded aggregates. In Vitro DOX Release from PEG-POSS-(TPE)7@DOX Aggregates. The pH-dependent DOX release measurement was conducted as below: dispersed DOX-loaded PEG-POSS-(TPE)7 aggregates were added into a dialysis membrane tube (MW cutoff, 4 kDa), which was then incubated in 30 mL of PBS (pH 5.0 and 7.4) at 37 °C in a shaking water bath at rate of 90 rpm. The pH-dependent DOX release profiles were determined by measuring the UV-vis absorbance of the solutions at 480 nm. All DOX-release experiments were conducted in triplicate and the results were expressed as the average data with standard deviations. To determine the total


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loading of the drug, the DOX-loaded aggregated suspensions were freeze-dried and then dissolved in DMF again, and analyzed with fluorescence spectroscopy. A calibration curve was obtained using DOX/DMF solution with different DOX concentrations. For determining the amount of DOX release, calibration curves were running with DOX/PBS (pH 7.4, 100 mM) with different DOX concentrations. Release experiments were conducted in triplicate. The results were presented as the average ±standard deviation. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formulas: DLC (wt %) = (weight of loaded drug/weight of polymer) ×100% DLE (wt %) = (weight of loaded drug/weight of feeding drug) × 100% Cellular uptake and intracellular localization of MCF-7 Cells Incubated with Blank Nanoparticles and DOX-Loaded Polymeric Aggregates. MCF-7 cells were plated on microscope slides in a 96-well plate (5 × 104 cells/well) using Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. After incubation for 24 h, the cells were incubated with prescribed amounts of free DOX (10 μg/mL), PEG-POSS-(TPE)7 nanoparticles, and DOXloaded PEG-POSS-(TPE)7 aggregates (equivalent to 10 μg/mL DOX) for 1 and 3 h at 37 °C and 5% CO2. Then the culture medium was removed and the cells on microscope plates were washed three times with PBS. After fixing with 4% paraformaldehyde overnight, the MCF-7 cells were observed under a fluorescent microscope with excitation at 330 nm and a confocal laser scanning microscopy (CLSM) with excitation at 405 nm for TPE and 488 nm for DOX.


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CCK-8 Assay. The cytotoxicity of PEG-POSS-(TPE)7 nanoparticles and DOX-loaded PEGPOSS-(TPE)7 aggregates were studied by CCK-8 assay using MCF-7 cells. Cells were seeded onto a 96-well plate at a density of 1×104 cells per well in 180 μL of DMEM containing 10% fetal bovine serum (FBS) and further incubated for 24 h (37 °C, 5% CO2). The medium was replaced by 90 μL of fresh DMEM medium containing 10% FBS, and then 20 μL samples of various concentrations (2-10 mg/mL) of the micelle suspensions in PBS (pH 7.4) were added. The cells were incubated for another 24 h. After removal of the culture media from cell culture plates, 100 μL of fresh culture media and 10 μL of CCK-8 kit solutions were immediately added and homogeneously mixed and then incubated for 4 h in a CO2 incubator. Finally, 100 μL of reaction solutions were put into 96-well plate. The optical density of each well at 450 nm was read by a microplate reader. Cells cultured in DMEM medium containing 10% FBS (without exposure to nanoparticles) were used as controls. RESULTS AND DISCUSSION Synthesis and Characterization of the Amphiphilic Tadpole-Shaped PEG-POSS-(TPE)7 Polymers. In view of the preparation of tadpole-shaped PEG350-POSS-(TPE)7 polymer in our previous work,18 the PEG1900-POSS-(TPE)7 polymer with longer hydrophilic PEG chains was produced according to the similar synthetic pathway in Scheme S1. Figure 1A clearly presented the assignments of PEG1900-POSS-(TPE)7 polymer. The signals at δ 6.8–7.6 ppm belonged to the protons of TPE groups and the resonance peak of δ 8.1 ppm was attributed to the Schiff base bonds while the locations at δ 3.3 ppm and 3.5–4.1 ppm were assigned to PEG chain in the 1H NMR spectrum. The integration ratio (Ia/Im/In) was extremely close to 3:7:133, confirming the intact structures of well-defined PEG1900-POSS-(TPE)7 polymer.

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C NMR spectrum also gave

the distinct evidence to clarify the rigid framework. To further testify the polymeric architecture


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with unimodal distribution of molecular weight, GPC coupled with triple detector array was employed to estimate the uniformity of the tadpole-shaped polymers. Figure S1 showed that the molecular weights of two polymers (PEG350-POSS-(TPE)7 and PEG1900-POSS-(TPE)7) were ca. 6300 and 7100 with narrow PDIs (1.07 and 1.06), which were close to their calculated values of 6017 and 7567. The NMR and GPC results manifested the successful preparation of well-defined PEG-POSS-(TPE)7 polymers.

Figure 1. (A) 1H and 13C NMR spectra of the PEG1900-POSS-(TPE)7 polymer. (B) Fluorescent spectra of the PEG1900-POSS-(TPE)7 polymer in THF/water mixtures excited by 330 nm. The insets depicted the fluorescence photographs.


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AIE Effects of the PEG-POSS-(TPE)7 Polymers. Similar to the PEG350-POSS-(TPE)7 polymer with AIE feature, when an amount of water was added into the THF solution of PEG1900-POSS-(TPE)7 polymer, the obvious fluorescence property of PEG1900-POSS-(TPE)7 was observed with non-emission in the THF solution but an intense fluorescence at 490 nm after addition of water into THF solution (Figure 1B). A possible explanation is that intramolecular rotation, served as a relaxation channel for the excited state to decay, was active in the organic solution. However, the rotation in the aggregates was restricted severely due to their spatial conformation and steric hindrance from the rigid POSS units, which forcefully blocked the nonradiative path and activated radiative decay. With the increase of water fraction, the fluorescence intensity of PEG1900-POSS-(TPE)7 continuously intensified and reached its maximal values at 99% water contents, indicating the formation of AIE-active and tighter aggregates.


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Figure 2. (A) TEM images, (B) AFM (height) images, (C) DLS profiles and (D) fluorescence microscopes of (a) PEG350-POSS-(TPE)7 vesicles, (b) PEG1900-POSS-(TPE)7 micelles, (c) before and (d) after incubation of (a) and (b) at pH 5.0 PBS solutions for 4 h. Self-Assembly of Amphiphilic Tadpole-Shaped Polymers and pH-Triggered Disruption of the Assemblies. The tadpole-shaped PEG350-POSS-(TPE)7 polymer, due to a mount of rigid TPE groups and tadpole-shaped topology that effectively obstruct direct strong aggregation of POSS units, can self-assemble into vesicles in aqueous solution with the diameter of 83 nm (TEM, Figure 2A-a), 115 nm (AFM, Figure 2B-a) and 136 nm (DLS, Figure 2C-a).18 The dimensional variation originated from the collapsed shape and swollen state of these PEG-POSS(TPE)7 nanoparticles. Determined by the hydrophilic/hydrophobic ratio for self-assembly behaviors, when the length of hydrophilic PEG chains extended, PEG1900-POSS-(TPE)7 polymer generated spherical micelles as expected with the average size of 60 nm (TEM) in Figure 2A-b, 135 nm (AFM) in Figure 2B-b and 165 nm (DLS) in Figure 2C-b, suggesting the better water absorbency than PEG350-POSS-(TPE)7 vesicles. It’s noted that the large size by AFM may be attributed to the effect of AFM tip.19 Dissipative particle dynamics (DPD) simulation is currently recognized as the viable simulation approach that can be employed to intuitively study the macromolecular morphological expressions,20 which provided a visualized formation pathway of the self-assembled vesicles and micelles (Scheme S2 and Figure 3). Notably, due to the tightly packing together of rigid POSS units and TPE segments in hydrophobic sections through strong π–π stacking, hydrogen bonding and hydrophobic forces, the intramolecular rotation of TPE groups was restricted extremely in the limited space. As a result, the assembled nanoparticles emitted strong cyan-light in the fluorescence microscope images as shown in Figure 2D-a and b. Importantly, as a result of their appropriate diameters (less than 200 nm) that are favorable to


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keep lowered level of reticuloendothelial system uptake, minimal renal excretion and efficient EPR effects for passive tumor-targeting, these fluorescent nanoparticles can be utilized in creation of ‘visible’ nano drug vehicles to trace loading drugs and their distributions within the cells during delivery.

Figure 3. The snapshots showing the formation pathway of (A) vesicle and (b) micelle. The water beads are omitted for clarity. Schiff base bond is stable enough under physiological condition (pH 7.4) and alkaline solution but readily cleavable in the endosomal conditions (pH 5.0-6.0), so the self-assembled nanoparticles containing Schiff base linkages can be quickly decomposed at pH 5.0 solution. TEM images (Figure 2A-c and d), AFM images (Figure 2B-c and d) and DLS results (Figure 2Cc and d) definitely revealed that both polymeric vesicles and micelles were dissociated into many fragments after acid treatment for 4 h. The sharp recession of cyan light in Figure 2D-c and d also suggested the escaped TPE-CHO molecules from the polymeric backbone, demonstrating the acid-triggered cleavage of Schiff base bonds and dissociation of nanoparticles. It was noted that although the TPE-CHO possessed AIE effect in the aggregate state, the free molecules might have no capacity to achieve the critical aggregate state to qualify the criterion of AIE effect, and these incompactly accumulational TPE-CHO segments were nearly non-emissive in this system.


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Figure 4. Size changes of (A) PEG350-POSS-(TPE)7 vesicles and (B) PEG1900-POSS-(TPE)7 micelles in response to (a) pH 5.0 and (b) pH 7.4 at 37 oC for various treated time determined by DLS measurement. To systematic clarification of the biodegradation process, we further quantitated the different pH (5.0 and 7.4) at various treated time by DLS measurement. By manipulating pH value of the solution to 5.0 shown in Figure 4A-a, the gradual swelling and slight collapse of the PEG350POSS-(TPE)7 vesicles occurred in the first 1 h. After 2 h, the swelling degree obviously increased and the cracking particles formed corresponding to one peak with up to 390 nm and another peak with 8 nm. Then the size further increased into 1000 nm and 15 nm with the treatment of 4 h. Compared to the original dimension, the incremental size was attributed to the released free TPE-CHO molecules in accumulation together and the vesicular swelling on account of the continuously improved hydrophilicity. The shrunken size ascribed to the tangled polymeric chains escaped from partially destroyed micelles, which originated from the fracture of a few Schiff base bonds. With standing for the longer time (8 h), more and more polymeric chains were extricated from the aggregates while a little bigger aggregation was ultimately formed. As for the PEG1900-POSS-(TPE)7 micelles at pH 5.0 solution, the size change and acid


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decomposition process exhibited the similar trends (Figure 4B-a). However, without polymeric decomposition and crack, the micelles only fully swelled in the solution with the size increased from 165 to 630 nm as well as a broad distribution in the first 2 h. After incubation for 4 h, the protonation of Schiff base bonds triggered the micelles disintegrate into small particles (collapsed micelles and fractured polymeric chains) and large aggregations (accumulated TPECHO). Then the acid-induced degradation continued to evolve in the pH 5.0 solution for 8 h, presenting the similar particle size distributions as the above, which implied the completely cleavage of Schiff base bands and disassemble of aggregates in such conditions. In comparison, both the polymeric vesicles and micelles at pH 7.4 solutions basically preserved their structural integrities but with only a slight increase in hydrodynamic diameters (Figure 4A-b and 4B-b). The incremental sizes were attributed to the micellar and vesicular swelling and/or hydrolysis after a long period of time.

Figure 5 Change of fluorescence intensities of (A) PEG350-POSS-(TPE)7 vesicles and (B) PEG1900-POSS-(TPE)7 micelles in response to various pH values (5.0, 5.8, 7.4 and 10.0) for (a) 0.5 h and (b) 24 h incubation.


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In view of highly luminous emissions by PEG350-POSS-(TPE)7 vesicles and PEG1900-POSS(TPE)7 micelles in a qualified state of aggregation (Figure 2D-a and b), we took advantage of their AIE effects to verify the pH-triggered disassembly by fluorescence measurement. As a consequence, we treated these two PEG-POSS-(TPE)7 nanoparticles with various pH values (5.0, 5.8, 7.4 and 10) in PBS solutions at different times (0.5 and 24 h) to observe the degradation process. In the first 0.5 h, the fluorescence intensity of PEG350-POSS-(TPE)7 vesicles gradually dropped at pH 5.8 and precipitously declined at pH 5.0 due to the beginning cleavage of Schiff base, and basically maintained the original level at pH 7.4 and 10.0 in Figure 5A-a. After treatment for 24 h, Figure 5A-b showed that the fluorescence intensities sharply dropped down into 14% and 22% of initial level at pH 5.0 and 5.8 solutions, indicating the severe breakage of Schiff base and decomposition of assemblies. At pH 7.4 and 10.0 solutions, the fluorescence intensities still remained 66% and 73% of initial level. This tiny decrease further proved the weak hydrolysis of a few Schiff bases in a long incubation time, which was consistent with the DLS results (Figure 4A-b). As for the PEG1900-POSS-(TPE)7 micelles, the varied trend of fluorescent intensities in the initial 0.5 h at various pH solutions was similar to that of PEG350POSS-(TPE)7 vesicles. Whereas, when the micelles were incubated under the same conditions for 24 h, the fluorescent intensities kept 41% and 56% at pH 5.0 and 5.8 PBS solutions (Figure 5B-a), and 85% and 91% of its original stage in neutral and alkaline environments (Figure 5B-b). This distinction may be attributed to that the Schiff base of PEG1900-POSS-(TPE)7 micelles were more tightly restricted (at the interior core) than that of PEG350-POSS-(TPE)7 vesicles (in the vesicular wall) although two polymeric assemblies exhibited the similar degradation behaviors under pH 5.0 condition (Figure 4A-a and 4B-a). Therefore, when the nanoparticles suffered acid erosion with various damage degrees, the relatively tighter micelles presented better AIE activity


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and emitted higher fluorescence. Based on the above analysis, it is revealed that both the PEGPOSS-(TPE)7 nanoparticles could preserve architectural integrity at physiological or alkaline pH but quickly disassemble in the presence of acid conditions, which can be employed as a class of visually and intelligently responsive drug carrier for tracing intracellular drug delivery. Loading and In Vitro Release of DOX. On the basis of these pH-responsive nanocarriers, anticancer drugs (DOX) were encapsulated into the vesicles and micelles by dialysis method, respectively. Figure S2 showed that both the DOX-loaded aggregates basically displayed spherical micelles and the average diameters of PEG350-POSS-(TPE)7@DOX and PEG1900POSS-(TPE)7@DOX micelles measured by DLS were about 185 and 172 nm. The loading content in the vesicles was 8.4% with the loading efficiency to be 25.3% while that in the micelles was 9.5% with the loading efficiency to be 26.6%. The in vitro release of DOX molecules from the PEG-POSS-(TPE)7@DOX aggregates was investigated at 37 °C under pH 5.0 and 7.4 PBS solutions. As shown in Figure 6, a gradual release of below 30% of encapsulated DOX within 24 h were observed at pH 7.4 for both PEG350-POSS-(TPE)7 vesicles and PEG1900-POSS-(TPE)7 micelles, which indicated that the DOX-loaded aggregates could basically preserve the core-shell architectures at a physiological pH along with a slow diffusion with a sustained release in a long period. In comparison, when the DOX-loaded aggregates were dispersed in an acidic condition, the incorporated DOX molecules rapidly released into the solution arising from the breakage of Schiff base, exhibiting a higher release content with 70% in the first 2 h and up to 95% in 24 h for PEG350-POSS-(TPE)7@DOX and with 60% and 91% in 2 and 24 h for PEG1900-POSS-(TPE)7@DOX aggregates. The high drug release rate may ascribe to that once the TPE-CHO molecules were released from the polymers, the hydrophilicity of assemblies was significantly improved, and therefore the remarkable swelling property enhanced


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the permeability of assemblies so as to allow the rapid leaking of loaded DOX molecules into the solution. This pH change from extracellular environment (pH 7.4) to the endosomal compartments (pH 5.0) implied that the DOX-loaded aggregates would remain stable in blood circulation and enhance drug release in the process of intracellular trafficking.

Figure 6. pH-triggered release of DOX from PEG350-POSS-(TPE)7 @DOX and PEG1900-POSS(TPE)7 @DOX aggregates with various pH values (5.0 and 7.4) at 37 °C as a function of time. Cell imaging of blank nanoparticles. As an ideal nano drug vehicles, the carriers should be efficiently endocytosed by tumor cells. On account of the AIE feature of these PEG-POSS(TPE)7 nanoparticles, the confocal laser scanning microscopy (CLSM) was used to trace their internalization and intracellular distributions. As shown in Figure 7A and 7B, the blue fluorescence in the cytoplasm regions were clearly observed within 1 h, which implied the rapid endocytosis into the cells. With increasing the incubation time for 3 h, more PEG-POSS-(TPE)7 nanoparticles were internalized into the tumors, leading to the apparent enhancement of the fluorescent intensities in Figure 7C and 7D. All these results revealed that the fluorescent nanoparticles can be easily internalized by the tumor cells.


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Figure 7. Confocal laser scanning microscopy images of the MCF-7 cells after incubation with blank vesicles (A, C) and micelles (B, D) for 1 and 3 h with the excitation of 405 nm. a: bright field image, b: blue fluorescence image, and c: overlap of bright field and fluorescence images. Scale bars are 20 μm. Cellular Uptake and Intracellular Drug Release. To determine the drug-loaded aggregates internalized by tumors possessing effective therapeutic effects, the cellular uptake and intracellular drug release behaviors of DOX-loaded aggregates (Scheme 2) were investigated by fluorescent microscope using MCF-7 cells. To the best of our knowledge, the hydrophobicity of DOX molecules always led to the substantial aggregation induced quenching in the process of intracellular trafficking, which was difficult for in situ DOX transportation scenario post systemic administration. To overcome this intractable problem, AIE and FRET effects are ideal


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techniques for nano drug vehicles to show the drug release within the cells. TPE groups were served as the amplifier to promote in situ surveillance of DOX based on the effective FRET (Figure S3) between TPE (donor) and DOX (acceptor) in our previous work.18 As displayed in Figure S4, these drug-loaded nanoparticles exhibited intensely red brightness instead of blue luminescence within the cells at the excitation of 330 nm, which was beneficial for FRET effect that made DOX detection more efficient. The intracellular luminescence indicated the rapid internalization into cancer cells via the endocytosis process. Scheme 2. Activated Intracellular Drug Release from pH-Sensitive Biodegradable Nanoparticles.

To further intuitively observe the location of drug carriers and image the intracellular drug delivery, we employed the CLSM to trace the cellular uptake and intracellular release behaviors of the DOX-loaded aggregates. As shown in Figure 8A and 8B, blue and red fluorescence were directly observed within the MCF-7 cells after incubation for 1 h, suggesting that these DOXloaded aggregates were easily internalized by cells and concentrated in the endosome. Along with the prolonged incubation time (3 h), the intracellular fluorescence intensities tended to be


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stronger and the cells (cytoplasm and nuclei) exhibited a clearer imaging in Figure 8C and 8D. Especially, the red fluorescence of DOX was clearly observed in the nuclei while the blue fluorescence of the drug delivery system was mainly concentrated in the cytoplasm. These results indicated that the DOX-loaded PEG-POSS-(TPE)7 aggregates had been successfully internalized by tumor cells with efficient release of DOX from the nanoparticles and their further escape from the endosome/lysosome to the nucleus, revealing that the drug delivery system had potential biological imaging applications.

Figure 8. Confocal laser scanning microscopy images of the MCF-7 cells after incubation with (A, C) DOX-loaded PEG350-POSS-(TPE)7 and (B, D) DOX-loaded PEG1900-POSS-(TPE)7 aggregates for 1 and 3 h with the excitation of 405 nm for TPE and 488 nm for DOX. Scale bars are 20 μm.


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Figure 8. (A) Cytotoxicity of PEG-POSS-(TPE)7 nanoparticles to MCF-7 cells following 24 h incubation. (B) Viabilities of MCF-7 cells following 24 h incubation with PEG350-POSS(TPE)7@DOX aggregates, PEG1900-POSS-(TPE)7@DOX aggregates and free DOX as a function of DOX dosages. All the data were presented as the average ±standard deviation. In vitro cell viability assay. The biocompatibility of the nanoparticles is a key issue in the drug delivery system. The in vitro cytotoxicity to MCF-7 cells of the nanoparticles was determined by CCK-8 assay. As seen in Figure 8A, both PEG-POSS-(TPE)7 assemblies were nontoxic to MCF-7 cells due to their quick degradation capacity in the presence of acidic conditions. The cell viabilities were more than 90% until a tested concentration of 1.0 mg/mL,


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demonstrating excellent biocompatibility of these biodegradable nanoparticles. However, the DOX-loaded aggregates displayed high efficiency of antitumor activity toward MCF-7 cells after incubation for 24 h. Figure 8B showed that the viability of MCF-7 cells depended on the DOX concentration. The DOX-loaded aggregates had a similar toxicity as free DOX in a low concentration. In a high concentration, DOX molecules could diffuse into cells rapidly, whereas the DOX-loaded aggregates had to be endocytosed to locate at the cells, which made free DOX molecules quicker than DOX-loaded aggregates in the internalization process, and thus displaying high efficiency in cancer cell inhibition. These results revealed that these biodegradable nanoparticles possessed an excellent biocompatibility and the DOX-loaded PEGPOSS-(TPE)7 aggregates showed a good antitumor capacity.

CONCLUSION In summary, we synthesized a kind of tadpole-shaped PEG-POSS-(TPE)7 polymers with pHresponsive properties and AIE features for tracing intracellular anticancer drug delivery. By adjusting the hydrophilic PEG length, these amphiphilic polymers self-assembled into fluorescent vesicles and micelles based on the significant AIE effects in aqueous solutions. Incorporation of Schiff base into the polymeric architectures furnished the fluorescent PEGPOSS-(TPE)7 nanoparticles with pH responsiveness that maintained the structural integrity at neutral or alkaline conditions and quickly disassembled in acidic conditions. After encapsulation of anticancer drug, PEG-POSS-(TPE)7@DOX aggregates exhibited pH-sensitive behaviors that rapidly and thoroughly release drug at the endo/lysosomal environments. Cytotoxicity assay indicated a fast internalization and a high cellular proliferation inhibition to MCF-7 cells.


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Notably, the intracellular distribution of the drug delivery system was observed directly using fluorescence microscopy with the help of efficient energy transfer from TPE donors to encapsulated DOX acceptors, exhibiting better intracellular imaging and self-localization property. Thus, we believe that this new type of proposed fluorescent system will provide an intriguing platform for targeted delivery with appreciable surveillance capacity, allowing in situ observing delivery scenario from the subcellular level to the range of the whole living body. ASSOCIATED CONTENT Supporting Information Experimental details and supporting figures were supplemented in Supporting Information. The material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT


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We acknowledge MOST (2014CB932200 and 2014BAI11B04), ‘Young Thousand Talents Program’ and NSFC (21504096, 51573195, 21174147 and 21474115) for financial support. REFERENCES (1) (a) Nel, A.; Xia, T.; Madler, L.; Li, N. Science 2006, 311, 622-627. (b) Zhang, X. L.; Huang, Y. X.; Ghazwani, M.; Zhang, P.; Li, J.; Thorne, S. H.; Li, S. ACS Macro Lett. 2015, 4, 620−623. (c) Laga, R.; Janouskova, O.; Ulbrich, K.; Pola, R.; Blazkova, J.; Filippov, S. K.; Etrych, T.; Pechar, M. Biomacromolecules 2015, 16, 2493−2505. (d) Hu, X. L.; Hu, J. M.; Tian, J.; Ge, Z. S.; Zhang, G. Y.; Luo, K. F.; Liu, S. Y. J. Am. Chem. Soc. 2013, 135, 17617−17629. (e) Nie, C. Y.; Wang, B.; Zhang, J. Y.; Cheng, Y. Q.; Lv, F. T.; Liu, L. B.; Wang, S. Small 2015, 11, 2555−2563. (2) (a) Zhou, Z. X.; Ma, X. P.; Murphy, C. J.; Jin, E. L.; Sun, Q. H.; Shen, Y. Q.; Van Kirk, E. A.; Murdoch, W. J. Angew. Chem., Int. Ed. 2014, 53, 10949−10955. (b) Duan, Q. P.; Cao, Y.; Li, Y.; Hu, X. Y.; Xiao, T. X.; Lin, C.; Pan, Y.; Wang, L. Y. J. Am. Chem. Soc. 2013, 135, 10542−10549. (c) Gupta, R.; Shea, J.; Scafe, C.; Shurlygina, A.; Rapoport, N. J. Control. Release 2015, 212, 70−77. (3) (a) Tahara, Y.; Akiyoshi, K. Adv. Drug Delivery Rev. 2015, 95, 65−76. (b) Ishii, S.; Kaneko, J.; Nagasaki, Y. Macromolecules 2015, 48, 3088−3094. (c) Kim, I.; Choi, J. S.; Lee, S.; Byeon, H. J.; Lee, E. S.; Shin, B. S.; Choi, H. G.; Lee, K. C.; Youn, Y. S. J. Control. Release 2015, 214, 30−39. (d) Cheng, C.; Tang, M. C.; Wu, C. S.; Simon, T.; Ko, F. H. ACS Appl. Mater. Interfaces 2015, 7, 19306−19315. (4) (a) Chen, W.; Meng, F. H.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z. J. Control. Release 2015, 210, 125−133. (b) Gao, X.; Wang, S. M.; Wang, B. L.; Deng, S. Y.; Liu, X. X.; Zhang, X. N.; Luo, L. L.; Fan, R. R.; Xiang, M. L.; You, C.; Wei, Y. Q.; Qian, Z. Y.; Guo, G. Biomaterials 2015, 53, 646−658. (c) Li, Y. L.; Zhu, L.; Liu, Z. Z.; Cheng, R.; Meng, F. H.; Cui, J. H.; Ji, S. J.; Zhong, Z. Y. Angew. Chem., Int. Ed. 2009, 48, 9914−9918. (5) (a) Jaskula-Sztul, R.; Xu, W. J.; Chen, G. J.; Harrison, A.; Dammalapati, A.; Nair, R.; Cheng, Y. Q.; Gong, S. Q.; Chen, H. Biomaterials 2016, 91, 1−10. (b) Liu, J.; Wei, T.; Zhao, J.; Huang, Y. Y.; Deng, H.; Kumar, A.; Wang, C. X.; Liang, Z. C.; Ma, X. W.; Liang, X. J. Biomaterials 2016, 91, 44−56. (c) Yang, Y. L.; Zhang, J. Y.; Liu, Z. Z.; Lin, Q. N.; Liu, X. L.; Bao, C. Y.; Wang, Y.; Zhu, L. Y. Adv. Mater. 2016, 28, 2724−2730. (6) (a) Rosler, A.; Vandermeulen, G. W. M.; Klok, H. A. Adv. Drug Delivery Rev. 2012, 64, 270−279. (b) Owen, S. C.; Chan, D. P. Y.; Shoichet, M. S. Nano Today 2012, 7, 53−65. (c) Wang, Z.; Ma, G. L.; Zhang, J.; Lin, W. F.; Ji, F. Q.; Bernards, M. T.; Chen, S. F. Langmuir 2014, 30, 3764−3774. (7) (a) Wu, X. M.; Zhu, W. H. Chem. Soc. Rev. 2015, 44, 4179−4184. (b) Shin, T. H.; Choi, Y.; Kim, S.; Cheon, J. Chem. Soc. Rev. 2015, 44, 4501−4516. (c) Hsiao, W. W. W.; Hui, Y. Y.; Tsai, P. C.; Chang, H. C. Acc. Chem. Res. 2016, 49, 400−407. (d) Stirland, D. L.; Matsumoto, Y.; Toh, K.; Kataoka, K.; Bae, Y. H. J. Control. Release 2016, 227, 38−44. (8) (a) Zou, Z.; He, D. G.; Cai, L. L.; He, X. X.; Wang, K. M.; Yang, X.; Li, L. L.; Li, S. Q.; Su, X. Y. ACS Appl. Mater. Interfaces 2015, 8, 8358−8366. (b) Feng, L. Z.; Gao, M.; Tao, D. L.; Chen, Q.; Wang, H. R.; Dong, Z. L.; Chen, M. W.; Liu, Z. Adv. Funct. Mater. 2016, 26, 2207−2217. (c) Lin, C. J.; Kuan, C. H.; Wang, L. W.; Wu, H. C.; Chen, Y.; Chang, C. W.;


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Title: Fabrication of pH-responsive nanoparticles with an AIE feature for imaging intracellular drug delivery

The biodegradable nanoparticles, assembled by the tadpole-shaped polymers, possess pHresponse and AIE feature for tracing intracellular drug delivery.


Fabrication of pH-Responsive Nanoparticles with an AIE Feature for

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