Two-Photon-Induced Charge-Variable Conjugated Polyelectrolyte

Mar 12, 2019 - Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics Science ...
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Two-Photon-Induced Charge-Variable Conjugated Polyelectrolyte Brushes for Effective Gene Silencing Hui Zhao,† Haojie Tao,† Wenbo Hu,‡ Xiaofei Miao,† Yufu Tang,† Tingchao He,§ Junzi Li,§ Qi Wang,‡ Lihong Guo,‡ Xiaomei Lu,‡ Wei Huang,‡,⊥ and Quli Fan*,†

ACS Appl. Bio Mater. Downloaded from pubs.acs.org by TULANE UNIV on 03/22/19. For personal use only.

†

Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China ‡ Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China § Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics Science & Technology, Shenzhen University, Shenzhen 518060, China ⊥ Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), Xi’an 710072, China S Supporting Information *

ABSTRACT: Cationic conjugated polyelectrolytes can absorb negatively charged small interfering RNA (siRNA) and also visualize the cellular internalization of siRNA, which thus have been extensively explored as siRNA carriers. However, their low charge density cannot afford a high carrying capability, severely impeding gene transfection efficiency. Moreover, the intracellular controlled release of siRNA is another factor that limits the widespread use of siRNA therapeutics. Herein, we present a novel twophoton-induced charge-variable conjugated polyelectrolyte brush as an efficient siRNA carrier. This cationic conjugated polyelectrolyte brush (PPENBr-ONB) with densely cationic charges produces remarkable carrying capability with siRNA. In addition, PPENBr-ONB with large two-photon absorption (TPA) cross-section represents effective fluorescence resonance energy transfer (FRET) to photoresponsive side chain with 720 nm illumination for two-photon-induced photolysis. Hence, the charge transformation of the photoresponsive side chain from cations to zwitterions would remarkably elevate siRNA release. The obtained PPENBr-ONB shows considerable fluorescence quantum yields (0.16) in aqueous solution, sufficient to serve as a reporter for cellular imaging. Agarose gel electrophoresis experiments indicate that PPENBr-ONB exhibit excellent siRNA-loading capacity (1 mol PPENBr-ONB to more than 20 mol siRNA). Furthermore, PPENBr-ONB with large TPA cross-section (1.47 × 105 GM) exhibits promoted siRNA release (78%) under 720 nm illumination. In vitro experiment shows that PPENBr-ONB/siRNA complex could efficaciously knock out of targeted Plk1 mRNA to 24.7% under 720 nm illumination for 1 h. This two-photon excitation siRNA carrier offers an efficacious strategy for the exploitation of photo controlled gene delivery system. KEYWORDS: two-photon absorption, charge-variable, conjugated polyelectrolyte brush, siRNA therapeutics

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INTRODUCTION Small interfering RNA (siRNA) has proven to be a therapeutic agent with bright prospect due to its highly efficient roles in disease-related genes in a sequence specific manner for biomedical research.1,2 A major limitation for an effective siRNA therapy was that naked siRNA was susceptible to degradation by nucleases and difficult to diffuse across cell membranes due to its highly anionic charge.3 To date, a variety of siRNA-carriers have been developed for the purpose of effective siRNA delivery such as polycations, inorganic nanoparticles, lipids, dendrimers, and peptides.1,4−7 Among them, water-soluble cationic conjugated polyelectrolytes have become one of the most commonly used strategies for delivery of siRNA since cationic side chains with multiple binding site can interact with negatively charged siRNA. Furthermore, © XXXX American Chemical Society

water-soluble cationic conjugated polyelectrolytes exhibited efficacious photoinduced signal transduction and the ability to resist photobleaching, making it possible for them to visualize the siRNA delivery process.8,9 Nevertheless, the further application of conjugated polyelectrolytes was still impeded. On the one hand, conjugated polyelectrolytes had a strong tendency to aggregate in aqueous solution, which brought about serious fluorescent quenching.10−12 On the other hand, the miserable carrying capability displayed a major bottleneck for siRNA delivery.4,8,9 Therefore, the development of Received: January 23, 2019 Accepted: March 12, 2019 Published: March 12, 2019 A

DOI: 10.1021/acsabm.9b00059 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Scheme 1. Reaction Route of Two-Photon-Induced Charge-Variable Polyelectrolyte Brush (PPENBr-ONB) and Its Photolysis (Top)a

a

Schematic representation of two-photon-induced charge-variable PPENBr-ONB for siRNA delivery and release.

energy transfer process.7,22 While the heavy metal of rare-earth may have latent chronic toxicity for biomedicine applications, in recent years, two-photon excitation (TPE) by means of absorbing two near-infrared (NIR) photons synchronously has emerged as a popular tool for remotely drug release due to its intrinsic 3D resolution, improved penetration depth, and reduced out-of focus photo damage.23,24 Unfortunately, the photoreactions induced by TPE were commonly inefficient as a result of low two-photon absorption (TPA) cross-section of most classical photodegradable groups. One alternative strategy was combining the photodegradable group with a two-photon absorber, suitable for transfer its excitation energy to photodegradable group in the NIR region.25,26 As an energy donor, the two-photon absorber must have sufficiently large TPA cross-section to ensure photodegradable efficiency. Therefore, the construction of a TPA-based photoresponsive system with large TPA cross-section was of considerable significance for promoted siRNA release.

efficacious system for siRNA delivery based on conjugated polyelectrolytes remained a big challenge. In addition, the intracellular controlled release of siRNA was another intractable problem for the widespread use of siRNA therapeutics. Cationic polymers were propitious to bind with siRNA; nevertheless, their strong electrostatic interaction will prevent siRNA release simultaneously.5 So far, several strategies have been established to promote siRNA release on the basis of low pH, enzyme, photo, reduction, and temperature.13−16 In the midst of them, photoresponsive strategy represented a unique avenue to achieve controlled release of siRNA in a highly spatial and temporal precision. However, most current photoresponsive systems were susceptible to UV irradiation, which would largely impede their further applications due to the shallow penetration depth of UV light and its detrimental high energy to biological tissue.17−21 To overcome this obstacle, upconversion nanoparticles (UCNPs) have been widely utilized to excite photodegradation as NIR light capturing material through B

DOI: 10.1021/acsabm.9b00059 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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drop by drop in 20 min. Then the solution was agitated for 12 h. The solution was then filtrated and evaporated on rotary evaporator. The product was depurated by silica gel chromatography (petroleum ether/ethyl acetate, 8:1) to receive the pure sample as light yellow solid (1.41 g, 84% yield). 1H NMR (CDCl3, 400 MHz, δ): 7.73−7.80 (s, 1H), 7.09−7.17(s, 1H), 5.62−5.69 (m, 2H), 4.16−4.23 (s, 2H), 3.94−4.07 (m, 6H). 13C NMR (CDCl3, 100 MHz, δ): 166.5, 153.7, 148.4, 139.8, 126.2, 110.1, 108.2, 64.5, 56.5, 40.7. GC−MS (m/z): 332.9 (M+). Synthesis of Macroinitiator. The conjugated polymer PPE was synthesized according to our previous report.26 The number-average molecular weight (Mn) of PPE was measured to be 5722 with a polydispersity index of 1.54. 1H NMR (CDCl3, 400 MHz, δ): 7.61− 7.74 (m), 7.42−7.57 (m), 7.10−7.16 (s), 4.16−4.26 (m), 4.11−4.16 (m), 3.86−3.96 (m), 2.02−2.15 (m). PPE (100 mg, 0.19 mmol) was then added into a 50 mL Schlenk flask, and the flask was eliminated and backfilled with argon three times. Then dry triethylamine (5 mL) and THF (10 mL) were syringed into the mixture. The solution was cooled to 0 °C, and then 2-bromoisobutyryl bromide (0.24 mL, 1.9 mmol) was added drop by drop. The reaction was then stirred for 3 h. The solvents were concentrated, and the product was precipitated into acetone and filtered. The filtrate was washed with water several times to remove the triethylamine salts. The precipitate was gathered and dehydrated to obtain the ATRP macroinitiator as yellow powder. 1 H NMR (CDCl3, 400 MHz, δ): 7.69−7.72 (m), 7.47−7.55 (m), 7.01−7.14 (br), 4.39−4.52 (br), 4.08−4.27 (m), 2.16−2.30 (br), 1.85−1.99 (s). Synthesis of PPEN. PPEN was synthesized as per our previous report.27 Macroinitiator (96 mg) and CuBr (20 mg) were mixed into a 50 mL Schlenk tube. To remove moisture and air, the tube was vacuumized and filled in nitrogen, and then o-dichlorobenzene (4 mL) and HMTETA (52 μL) were added. The tube was heated to 90 °C. Then DMAEMA (6 mL) was rapidly added into the reaction tube to implement polymerization. After the reaction was completed, the solution in the tube was filtrated and then precipitated in n-hexane to obtain PPEN as light yellow solid. GPC (80478, polydispersity index of 1.36), 1H NMR (CDCl3, 400 MHz, δ): 4.01−4.17 (s), 2.73−3.03 (s), 2.50−2.70 (s), 2.23−2.41 (s), 1.73−2.12 (d, J = 32.9 Hz), 0.99− 1.50 (s), 0.77−0.98 (s). Synthesis of PPENBr-ONB. In a 100 mL round-bottom flask, PPEN (0.5 g) was dissolved in THF (12 mL). 4,5-Dimethoxy-2nitrobenzyl 2-bromoacetate (0.15 g) was then added. The solution was agitated at 50 °C for 12 h, and then some water was added. When the precipitation disappeared, the solution was agitated for another 24 h and then depurated through dialysis with ultrapure water for 3 days by utilizing dialysis membrane (5.0 kDa) to discard low-molecularweight parts. With freeze-drying, the product of PPENBr-ONB with yellow solid was received. GPC (83418, polydispersity index of 1.29), 1 H NMR (D2O, 400 MHz, δ): 7.46−7.14 (m), 4.54−4.17 (s), 4.15− 3.74 (d, J = 17.8 Hz), 3.53−3.37 (s), 3.37−3.02 (d, J = 21.7 Hz), 2.96−2.71 (s), 2.15−1.72 (br, J = 90.4 Hz), 1.14−0.64 (m, J = 62.2 Hz). Measurement of Quantum Yield. The quantum yield of PPENBr-ONB was monitored using quinine sulfate as a reference (55%, 0.1 M H2SO4 as solvent).28 Configured quinine sulfate solution in 0.1 M H2SO4 and adjust the concentration to ensure that the absorbance of quinine sulfate was less than 0.1 to decrease reabsorption effects. Then adjust the PPENBr-ONB aqueous solution to the same absorbance with quinine sulfate at 320 nm. The fluorescence spectra of quinine sulfate and PPENBr-ONB were determined under the same condition with 320 nm excitation. Then the integrated area of the fluorescence spectra was utilized to calculate the quantum yields (QY) of PPENBr-ONB by utilizing the following equation:

Herein, we report the two-photon-induced charge-variable conjugated polyelectrolyte brushes (PPENBr-ONB) for siRNA delivery and effective gene silencing (Scheme 1). In our design, the cationic conjugated polyelectrolyte brushes were constitute of rigid backbone (poly(phenylene ethynylene)) (PPE) and flexible side chains (poly(methyl methacrylate) derivative) with high density. Further, photodegradable chromophore onitrobenzyl (ONB) was added onto conjugated polyelectrolyte brushes by quaternary ammoniation for promoting the release of siRNA via two-photon-induced photodegradation. On one hand, the high density cationic charges of the brush side chain produces outstanding carrying capability with siRNA. On the other hand, PPE fluorophore with large two-photon crosssection was not only used as a reporter for cellular imaging, but also as a two-photon light capturing material to absorb energy. Subsequently, the electronic excitation of PPE was transferred to ONB, breaking it under 720 nm illumination for promoting siRNA release via charge change of the side chain from cations to zwitterions. In this work, the as prepared PPENBr-ONB presents considerable fluorescence quantum yields (0.16) in water and outstanding siRNA carrying capacity (1 mol PPENBr-ONB to more than 20 mol siRNA). Furthermore, PPENBr-ONB with large TPA cross-section (1.47 × 105 GM) exhibits promoted siRNA release (78%) and could efficaciously knock out of targeted Plk1 mRNA to 24.7% with 720 nm illumination for 1 h. In a word, this siRNA delivery system based on two-photon-triggered conjugated polyelectrolyte brushes opens new perspectives for the extension of siRNAbased therapeutics.

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MATERIALS AND METHODS

Materials and Instruments. The cy3-labeled siRNA and Plk1 were purchased from Guangzhou RiboBio. Unless specified, other reagents for organic synthesis were bought from Sigma-Aldrich. 1H and 13C NMR spectra were detected on a Bruker Ultra Shield Plus 400 MHz spectrometer (1H: 400 MHz, 13C: 100 MHz). The absorption and fluorescence spectra were detected by Shimadzu UV3600 UV−vis NIR spectrophotometer and a RF-5301PC spectrofluorophotometer, respectively. PL quantum yields (QYs) were determined utilizing the quinine sulfate as reference, with the quantum yield of 55%. All the spectral experiments were implemented at room temperature. Gas chromatography−mass spectrometry was implemented using a Shimadzu GC−MS-QP 2010 Plus mass spectrometer. Gel permeation chromatography (GPC) analysis was detected on Shimadzu LC-20A using polystyrene as the standard and THF as an eluent. JEOL JEM-2100 transmission electron microscope (TEM) was utilized to determine the TEM images manipulating at an acceleration voltage of 100 kV. Zeta potentials were detected by ZetaPALS (Brookhaven Instruments Corp). The hydrodynamic sizes were measured through dynamic light scattering (DLS) by a 90 Plus particle size analyzer (Brookhaven Instruments). Microplate reader (BioTek, PowerWave XS/XS2, U.S.) was utilized to performed MTT assay. Two-photon tests originated from a Ti:sapphire laser regulated to 720 nm (∼3 W), which corresponded to approximately 1% (∼30 mW at 720 nm) average power in the focal plane. The lifetime was determined by Edinburgh FLSP920 fluorescence spectrophotometer assembled with a pulsed semiconductor light source. The gene silencing efficiency was measured using the DA7600 fluorescence quantitative PCR circulation apparatus (Zhongshan Daan, China). Cellular imaging was received by a confocal laser scanning microscope (CLSM) (Leica, TCS SP5, Germany). Synthesis of 4,5-Dimethoxy-2-nitrobenzyl 2-bromoacetate. 4-Dimethylaminopyridine (DMAP) (0.062 g, 0.5 mmol) and (4,5dimethoxy-2-nitrophenyl)methanol (1.07 g, 5 mmol) were dissolved in anhydrous THF (20 mL) and then cooled to 0 °C in nitrogen atmosphere. 2-Bromoacetyl chloride (0.94 g, 6 mmol) was mixed

QY2 = QY1

2 I2 A1 η2 I1 A 2 η12

(1)

Where I represented the integrated fluorescence intensity, η represented the refractive index for the solvent, and A indicated the C

DOI: 10.1021/acsabm.9b00059 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 1. (a) TEM and DLS (inset) of PPENBr-ONB. (b) TEM and DLS (inset) of PPENBr-ONB/siRNA complex. (c) Zeta potential of PPENBr-ONB and PPENBr-ONB/siRNA complex. Cell Viability Assay. Cytotoxicity of PPENBr-ONB/siRNA was detected in HeLa cells by MTT assay. To determine the chemotoxicity of PPENBr-ONB/siRNA, HeLa cells were cultured in 96-well plate (0.5 × 104 cells per well) in DMEM for 24 h. After that, the cells were cultivated with a certain amount of PPENBr-ONB/siRNA complex for 4 h. The concentration of PPENBr-ONB was 40 nM, and the concentrations of siRNA were 0, 20, 50, 100, 200, and 400 nM, respectively. The cells without treatment were used as a control. The cell viability was implemented by MTT assay after 48 h incubation. SiRNA Release Experiments. SiRNA release of PPENBr-ONB/ siRNA complexes with or without 720 nm illumination can be examined using dialysis method. PPENBr-ONB/siRNA complexes solution (200 μL) without 720 nm illumination was dialyzed with dialysis bag (10 kDa), which was located in 10 mL of 10−2 mol/L PBS. PPENBr-ONB/siRNA complexes with 720 nm illumination for 1 h were also managed under the same condition. In consideration of the subsequent research of transfection efficiency, the proportion of PPENBr-ONB to siRNA was adjusted to 1:20. After 0.5, 1, 2, 4, 6, 8, and 24 h, respectively, PBS was displaced by 10 mL of pure PBS, and the solution withdrawn was utilized to detect the concentration of siRNA by determining its absorption at 260 nm. The released siRNA can be evaluated in comparison with the standard concentration curve. The loading amount of PPENBr-ONB to siRNA was set as 100%. Then the accumulative release amount of siRNA can be monitored. As represented before, FRET between PPENBr-ONB and siRNA-cy3 molecule will generate when they combined together. The FRET between PPENBr-ONB and siRNA will be weakened if siRNA released from the complex. Thus, the cell imaging can also be utilized to visualize the release of siRNA intracellularly. HeLa cells were first cultured with DMEM medium containing 40 nM PPENBr-ONB and 200 nM siRNA-cy3, which were then treated with 720 nm illumination for 1 h. Confocal microscope was utilized to take cellular images, and the siRNA release was visualized through the fluorescence changes of PPENBr-ONB and siRNA-cy3. SiPlk1 Transfection and Real-Time Fluorescent Quantitative Polymerase Chain Reaction (qRT-PCR). HeLa cells were cultured in confocal dish at 4 × 105 cells per well in complete DMEM medium in incubator for 24 h. Afterward, the solution was substituted with culture medium containing PPENBr-ONB/siPlk1 complexes with 720 nm illumination for different times and then incubated for 4 h. The sequences of SiPlk1 were the same as our previous report.32 The concentration of siPlk1 and PPENBr-ONB in the culture medium was 120 nM and 50 nM, respectively. After that, the medium was displaced with pure DMEM for 24 h. The relative Plk1 mRNA levels were evaluated by qRT-PCR in which the RNA of transfected cells was separated by TRIzol reagent. Then the RNA was transcribed into cDNA. After that, cDNA targeting Plk1 and glycer-aldehyde 3phosphate dehydrogenase (GAPDH) was exposed to qRT-PCR analysis. The outcomes were proposed as the multiple difference in Plk1 expression normalized to GAPDH as the reference and relative to the controlled group without treatment. The GAPDH and Plk1 primers and the parameters of PCR were the same as previously reported.32 The relative amount of target gene mRNA was normalized to GAPDH mRNA. Specificity was verified by melt curve analysis.

absorbance at the excitation wavelength. Numbers 1 and 2 represented the quinine sulfate and PPENBr-ONB, respectively. Measurement of TPA Cross-Section. TPA cross-section (δ) of PPENBr-ONB was measured by the two-photon induced fluorescence method. Rhodamine 6G in ethanol was used as in ref 29. The twophoton cross-section of Rhodamine 6G at different wavelength can be found in previous report.30 The quantum yield of Rhodamine 6G in ethanol was 95%.31 A Ti:sapphire system that produced 100 fs (HW1/e) pulses at a repetition of 80 MHz was used to excite the two-photon fluorescence. The two-photon induced fluorescence intensity of Rhodamine 6G and PPENBr-ONB was tested under the same experimental conditions. The TPA cross-section (δ) of PPENBr-ONB can be calculated utilizing the following equation: δ2 = δ1

F2 φ1 c1 n2 F1 φ2 c 2 n1

(2)

where δ represented the TPA cross-section, F was the fluorescence intensity, φ was the fluorescence quantum yield, c was the molar concentration, and n indicated the refractive index. Numbers 1 and 2 represented the Rhodamine 6G and PPENBr-ONB, respectively. Agarose Gel Electrophoresis Experiments. To determine the loading efficiency of PPENBr-ONB to siRNA, PPENBr-ONB loaded siRNA with different molar ratios was established in TAE buffer at 170 V for 15 min by 0.6% agarose gel. SiRNA was colored by GelRed. Then a digital camera was used to take photos of the gel with the illumination of UV light. To demonstrate the steadiness of PPENBrONB/siRNA toward ribonuclease (RNase A), PPENBr-ONB loaded with siRNA was treated with RNase A and siRNA alone was used as a control. Loading Efficiency of PPENBr-ONB to siRNA. The loading efficiency of PPENBr-ONB to siRNA was assessed by agarose gel electrophoresis assay. When the amount of siRNA increased, the surface charges of PPENBr-ONB were neutralized, which resulted in the reduced mobility of PPENBr-ONB/siRNA complexes. When the amount of siRNA overloaded, free siRNA that cannot be loaded by PPENBr-ONB appeared. Hence, the highest amount of siRNA that can be loaded by PPENBr-ONB can be taken as the loading efficiency of PPENBr-ONB to siRNA. Cellular Internalization of PPENBr-ONB/siRNA. The cellular internalization of PPENBr-ONB/siRNA complexes in HeLa cells was determined. The cells were first incubated with DMEM medium in incubator for 24 h. After that, the medium was substituted with culture medium containing 40 nM PPENBr-ONB and 20 μM siRNAcy3. The cells were washed two times with phosphate buffer solution (PBS) after incubation for 4 h. CLSM was utilized to visualize the cellular internalization of PPENBr-ONB/siRNA. Flow analysis was also determined to further conform the cell uptake of PPENBr-ONB/ siRNA. HeLa cells were seeded in six-well plates containing DMEM medium. The PPENBr-ONB/siRNA complex was added to the culture and incubated for 4 h, and the cells incubated with siRNA-cy3 alone or without treatment were set as controls. After digesting and washing with PBS, the cells were dispersed in 1 mL of PBS, and the cellular uptake of PPENBr-ONB/siRNA was measured via flow cytometry. D

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Figure 2. (a) Normalized absorption spectrum of ONB (black) and emission spectrum of PPENBr-ONB (red) (λex = 350 nm). (b) Normalized absorption spectrum of PPENBr-ONB (red) and emission spectra of PPEN (blue), and PPENBr-ONB (black). (c) Fluorescence decay curve of PPEN and PPENBr-ONB with the excitation at 350 nm, monitored at 450 nm. (d) TPA cross-section of PPENBr-ONB at different wavelengths. (e) Two-photon fluorescence spectra of PPENBr-ONB with 720 nm irradiation. (f) Power dependence of the two-photon fluorescent intensity on the femtosecond laser.

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RESULTS AND DISCUSSION Characterization of PPENBr-ONB and PPENBr-ONB/ siRNA Complex. The positively charged brush-type polymer PPENBr-ONB was prepared via quaternary ammoniation of N-functionalized PPE (PPEN) using photoresponsive chromophore 4,5-dimethoxy-2-nitrobenzyl 2-bromoacetate as shown in Scheme S1. PPEN was synthesized in accordance with our previous report.33 PPEN was composed of a poly(phenylene ethynylene) backbone with the polymerization degree of 22 and multiple side chain of poly(2(dimethylamino)ethyl methacrylate) with the average monomer number of 20 per unit. From 1H NMR of PPENBr-ONB (Figure S4), the proportion of the peak areas of methyl hydrogen (N−CH3, 2.84 ppm) and nitro aromatic hydrogen (NO2−Ph-H, 7.30 ppm) indicated that the quaternization degree was about 54%. The PPENBr-ONB/siRNA complex was formed by mixing PPENBr-ONB and siRNA at different molar ratios in PBS and stirring for 30 min. Owing to the strong electrostatic interaction between siRNA and the cationic side chains of PPENBr-ONB with high density, the size and surface charges of PPENBr-ONB will be altered after loading with siRNA. TEM images of Figure 1a showed that the PPENBr-ONB formed nanoparticles with the size of 40 nm indicating the successful self-assembly of PPENBr-ONB in aqueous solution. After mixing with siRNA, the particle size was visibly enhanced to 60 nm, indicating the binding of siRNA to PPENBr-ONB nanoparticles (Figure 1b). The hydrodynamic radii of PPENBr-ONB and PPENBr-ONB/siRNA complex measured by dynamic light scattering (DLS) were determined to be 48 and 76 nm, which were larger than those in TEM (insets of Figure 1a,b). TEM and DLS also displayed observably welldistributed appearances of the nanoparticle, which will be conducive to form stable siRNA delivery system. Zeta potentials of PPENBr-ONB and PPENBr-ONB/siRNA complex were also measured. From Figure 1c, zeta potential of PPENBr-ONB nanoparticles was about 31.57 ± 0.80 mV in

aqueous solution. Such positive zeta potential provided favorable conditions for siRNA delivery. After mixing with siRNA, the potential of PPENBr-ONB/siRNA complex was reduced to 8.33 ± 1.25 mV, demonstrating the successful binding of siRNA to PPENBr-ONB nanoparticles. Optical Properties of PPENBr-ONB. From Figure 2a, the black and red line, respectively, represented the absorption spectrum of ONB and fluorescent spectrum of PPENBr-ONB in aqueous solution. The obvious overlap between the emission of PPENBr-ONB and absorption of ONB from 360 to 400 nm was a necessary condition for efficient energy transfer.34 The absorption of PPENBr-ONB was just the composition of the absorption spectra of ONB and PPEN, which demonstrated the successfully quaternary ammoniation of PPEN (Figure 2b). Besides, the emission of PPENBr-ONB showed a weak red shift compared with PPEN, suggesting a slight effect of aggregation of PPENBr-ONB in aqueous solution. To verify the energy transfer process between PPEN and ONB, time-resolved photoluminescence (TRPL) was performed to determine the emission lifetimes of PPEN and PPENBr-ONB at 450 nm. From Figure 2b, the fitting results of the average lifetimes of PPEN and PPENBr-ONB were 1.02 and 0.73 ns. The decreased lifetime of PPENBr-ONB can be attributed to the energy transfer between PPEN and ONB since FRET played the part of an extra energy decay channel.35,36 We can also estimate the energy transfer efficiency by the following equation to describe the energy transfer dynamics: E=1−

τ τ0

(3)

Where τ was the lifetime of the donor PPENBr-ONB with the acceptor ONB, and τ0 was the lifetime of PPEN without ONB.37 The energy transfer efficiency was computed to be 28.4% for this system. The fluorescence quantum yield (QY) of PPENBr-ONB in aqueous solution was 16%, determined using quinine sulfate in E

DOI: 10.1021/acsabm.9b00059 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 3. (a) Zeta potential changes of PPENBr-ONB with prolonged time of 720 nm light illumination. (b) 1H NMR of the product 4,5dimethoxy-2-nitrobenzyl aldehyde. (c) Absorbance spectra changes of the product 4,5-dimethoxy-2-nitrobenzyl aldehyde under 720 nm illumination.

4,5-dimethoxy-2-nitrobenzyl aldehyde can be seen clearly. Moreover, the absorption spectra of the dialysate after different illumination time were determined. From Figure 3c, obvious bands in 300−400 nm appeared after exposure to 720 nm laser, which further proved the formation of the photolysis product 4,5-dimethoxy-2-nitrobenzyl aldehyde. Binding Capacity of PPENBr-ONB with siRNA. Agarose gel electrophoresis assay was used to estimate the carring capacity of PPENBr-ONB to siRNA. From Figure 4, siRNA

0.1 M H2SO4. This value compared favorably to the corresponding PPE-based linear polyelectrolyte with a QY < 10%,38,39 suggesting that cationic brush was beneficial to yield PPE with high fluorescence QY for effective fluorescent chemical sensing. To characterize the two-photon excitation ability of PPENBr-ONB, its TPA cross-section was gauged using two-photon-induced fluorescence method. Rhodamine 6G was used as the reference. The details were shown in the Materials and Methods section. From Figure 2d, the TPA cross-section of PPENBr-ONB reached the maximum of 1.47 × 105 at 720 nm and decreased gradually with the wavelength increased. Hence, 720 nm illumination was selected to perform the following photolysis process. This value was comparable with the high-performance water-soluble inorganic two-photon fluorescence nanomaterial whose TPA cross-section was about 4.7 × 104 GM.40 Such a large TPA cross section enabled a great potential of PPENBr-ONB for two-photon-induced photodegradation. On the basis of the above results, two-photon fluorescence spectra of PPENBr-ONB were determined. From Figure 2e, PPENBr-ONB in aqueous solution exhibited strong fluorescence under 720 nm excitation. We also measured the relationship between the two-photon fluorescence intensity of PPENBr-ONB and the power of the femtosecond laser. The square dependence of fluorescence intensity to the power proved the TPA process (Figure 2f). Photolysis of PPENBr-ONB. To verify that the emission of PPENBr-ONB was efficacious to disrupt ONB, zeta potentials of PPENBr-ONB with prolonged time of 720 nm femtosecond laser illumination (ca. 30 mW) were determined. The subsequent illumination procedures were implemented in the same manner. As shown in Figure 3a, zeta potential of PPENBr-ONB was 31.57 ± 0.80 mV before illumination and then declined by degrees with the prolongation of irradiation time. With 720 nm illumination for 100 min, the zeta potential declined to 13 mV and the slope became smoothly with extended illumination time. The declining zeta potential mostly ascribed to the production of carboxyl anion owing to the photodegradation. To further confirm this photodegradation process, PPENBr-ONB was dialyzed using dialysis bag under 720 nm laser illumination. The molecular weight cutoff of the dialysis bag was 10 kDa so that the photolysis product would pass through the dialysis bag easily. Then 1H NMR spectra of the dialysate were determined and shown in Figure 3b. The signals at 3.99, 7.12, and 7.78 ppm, respectively, corresponding to the methyl hydrogen (O−CH3) and aromatic hydrogen (NO2−Ph-H) of the photolysis product

Figure 4. Agarose gel electrophoresis assays of PPENBr-ONB/siRNA complex with different molar ratios. Lane 1 represented siRNA only. Lanes 2−7 were PPENBr-ONB mixed with siRNA with different molar ratios (1:5, 1:10, 1:15, 1:20, 1:30, 1:40).

with negative charge moved toward the anode while PPENBrONB brush with positive charge would move toward the contrary direction. When the proportion of siRNA/PPENBrONB increased, the charges of PPENBr-ONB were neutralized by degree and moved toward the negative pole with weakened mobility. When the proportion of siRNA/PPENBr-ONB increased to 20:1, free siRNA emerged and moved toward the anode, which indicated that at least 20 mol siRNA could be efficaciously loaded by 1 mol PPENBr-ONB. The outstanding carrying capability of PPENBr-ONB to siRNA would decrease the applicaiton concentration of PPENBr-ONB and hence realize efficacious siRNA delivery system for gene silencing. We also utilized the agarose gel electrophoresis experiment to prove that the combination of siRNA and PPENBr-ONB can protect siRNA from enzymatic degradation. Figure S6 F

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Figure 5. (a) CLSM images of HeLa cells: (A) treated with siRNA-cy3; (B) treated with PPENBr-ONB; (C) treated with siRNA-cy3 and PPENBr-ONB. (b) Flow cytometry analysis of HeLa cells incubated with siRNA-cy3 or PPENBr/siRNA-cy3 complex. (c) siRNA release from PPENBr-ONB/siRNA in pH 7.4 and pH 5.0 with or without 720 nm illumination.

Figure 6. siRNA release from the complex intracellularly: (A) treated with PPENBr-ONB/siRNA-cy3 for 4 h without 720 nm illumination; (B) treated with PPENBr-ONB/siRNA-cy3 for another 1 h without 720 nm illumination; (C) treated with PPENBr-ONB/siRNA-cy3 for another 1 h with 720 nm illumination for 1 h. The first and second channels were measured at 420−480 and 580−620 nm, respectively, with 720 nm illumination, the third channel was the overlap of the first and second channel with the brightfield, the fourth channel was measured at 580−620 nm with 559 nm excitation, and the last channel was a combination of this channel, the first channel, and the brightfield.

ability of PPENBr-ONB would thus increase the intracellular lifetime of siRNA. In Vitro Delivery and Release of siRNA. Negatively charged siRNA was proved to be difficult for cellular uptake. Herein, PPENBr-ONB/siRNA complex was expected to improve siRNA cellular uptake. Before the utilization of this delivery system intracellularly, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was implemented

displayed that siRNA alone moved toward anode (lane 1) and were entirely decomposed when mixed with RNase A (lane 2), while siRNA combined with PPENBr-ONB moved toward negtive pole (lane 3) and kept perfectly when mixed with RNase A (lane 4). This phenomenon manifested that PPENBr-ONB/siRNA complex could efficaciously defend siRNA from RNase degradation and the well siRNA protection G

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Figure 7. Protein expression (top) and gene silencing efficiency (below) of PPENBr-ONB/siRNA delivery system.

to evaluate the cell viability. As shown in Figure S10, cells incubated with PPENBr-ONB/siRNA in different mole ratio (1:0, 1:0.5, 1:1.25, 1:2.5, 1:5, 1:10) kept high viability, which demonstrated well biocompatibility of PPENBr-ONB/siRNA complex. Then PPENBr-ONB/siRNA complex with mole ratio of 1:1.25 was utilized to transfect HeLa cells, and the cells incubated with siRNA-cy3 or PPENBr-ONB alone were set as controls. From Figure 5a, the second channel was collected under 720 nm illumination and the third channel was collected with illumination at 559 nm (Figure 5aA,B). The overlap of the blue fluorescence of PPENBr-ONB and red fluorescence of siRNA-cy3 indicated the outstanding improvement of siRNA internalization. Flow analysis was also determined to further confirm the cell uptake of PPENBr-ONB/siRNA. From Figure 5b, HeLa cells treated with PPENBr-ONB/siRNA complex showed significant increased fluorescence in comparison with the free siRNA. This phenomenon suggested that PPENBrONB can effectively deliver siRNA into cells. The releasing of siRNA was detected by detecting the siRNA concentration of the dialysate after different illumination time. From Figure 5c, the ratios of released siRNA to original carrying capacity of siRNA at pH 7.4 and pH 5.0 were, respectively, corresponding to the black and red line. The released siRNA with 720 nm illumination for 1 h at pH 7.4 were equivalent to the blue line. It was shown that approximately 45% siRNA was released from PPENBr-ONB at pH 5.0, which mainly ascribed to the protonation effect, while over 80% siRNA was released at pH 5.0 with 720 nm illumination for 1 h. This can be attributed to the lightswitchable polymer side chain, which could be cleaved from cationic to zwitterionic under 720 nm irradiation. The promoted siRNA intracellular release from PPENBr-ONB under 720 nm illunimation can also be visualized by CLSM

due to the distinct effects of FRET between blue PPENBrONB and red siRNA-cy3 molecules. Cell images of siRNA release with or without 720 nm illunination were displayed in Figure 6. The signals in the first and second channels showed that both of PPENBr-ONB and siRNA-cy3 emitted strong fluorescence with 720 nm excitation. Since the majority of siRNA were binding compactly with PPENBr-ONB through strong electrostatic interaction, the energy absorbed by PPENBr-ONB with 720 nm excitation can be effectively transferred to siRNA-cy3 and thus emitting red light produced by cy3. When the cells treated with PPENBr-ONB/siRNA for another 1 h, PPENBr-ONB still emitted strong blue fluorescence, while the red fluorescence by cy3 slightly decreased (Figure 6B). This is due to the partially release of siRNA from the complex after 1 h transfection. After 720 nm illumination for 1 h, red signals from cy3 almost disappeared in Figure 6C, which ascribed to the most release of siRNA from PPENBr-ONB/siRNA after 1 h illumination. The FRET between PPENBr-ONB and cy3 was severely weakened, while the red fluorescence of cy3 was lighted when the cells were irradiated at 559 nm. From Figure 6A, the well overlap of blue PPENBr-ONB with 720 nm excitation and red siRNA-cy3 with 559 nm excitation also showed that most of siRNA did not release. Yet the overlay image in Figure 6C indicated considerable discrepancy in the positions intracellularlly after 1 h illumination, which demonstrated the promoted siRNA release in cells. Hence, the FRET cellular imaging can be utilized to visulize the release process of siRNA intracellularlly. RNAi Efficiency. According to previous reports, Polo-like kinase 1(Plk1) played a crucial role in DNA replication and was overexpressed in many types of cancer cells.41 To evaluate the RNAi efficiency of PPENBr-ONB, PPENBr-ONB/siPlk1 were cultured with HeLa cells at 37 °C for 4 h. Different H

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and Information Displays, and Synergetic Innovation Center for Organic Electronics and Information Displays.

irradiation time was treated with them. We utilized qRT-PCR to monitor the targeted mRNA Plk1 expression level after culturing 20 h. Cells cultured with PBS were used as a control. From Figure 7, the relative Plk1 mRNA levels were almost unchanged when the cells were incubated with 50 nmol/L PPENBr-ONB alone. The relative Plk1 mRNA levels were decreased to 80% after the cells were treated with 50 nmol/L PPENBr-ONB and 120 nmol/L siPlk1, which was mainly due to the partly release of siRNA. After the cells were incubated with PPENBr-ONB/siPlk1 with 720 nm illumination, the Plk1 mRNA levels were dropped as the prolongation of illumination time. The RNAi effects seemed to be more efficacious and the gene knock down was achieved to 40% after 1 h illumination. This result showed the great validity of the platform for siRNA delivery and release.

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(1) Zhao, Y.; Wang, W.; Guo, S.; Wang, Y.; Miao, L.; Xiong, Y.; Huang, L. PolyMetformin Combines Carrier and Anticancer Activities for in vivo siRNA Delivery. Nat. Commun. 2016, 7, 11822−11830. (2) Xu, X.; Wu, J.; Liu, Y.; Yu, M.; Zhao, L.; Zhu, X.; Bhasin, S.; Li, Q.; Ha, E.; Shi, J.; Farokhzad, O. C. Ultra-Ph-Responsive and TumorPenetrating Nanoplatform for Targeted Sirna Delivery with Robust Anti-Cancer Efficacy. Angew. Chem., Int. Ed. 2016, 55, 7091−7094. (3) Yang, G.; Lv, F.; Wang, B.; Liu, L.; Yang, Q.; Wang, S. Multifunctional Non-Viral Delivery Systems Based on Conjugated Polymers. Macromol. Biosci. 2012, 12, 1600−1614. (4) Fang, H.; Guo, Z.; Lin, L.; Chen, J.; Sun, P.; Wu, J.; Xu, C.; Tian, H.; Chen, X. Molecular Strings Significantly Improved the Gene Transfection Efficiency of Polycations. J. Am. Chem. Soc. 2018, 140, 11992−12000. (5) Zhu, D.; Yan, H.; Liu, X.; Xiang, J.; Zhou, Z.; Tang, J.; Liu, X.; Shen, Y. Intracellularly Disintegratable Polysulfoniums for Efficient Gene Delivery. Adv. Funct. Mater. 2017, 27, 1606826. (6) Xiang, Y.; Oo, N. N. L.; Lee, J. P.; Li, Z.; Loh, X. J. Recent Development of Synthetic Nonviral Systems for Sustained Gene Delivery. Drug Discovery Today 2017, 22, 1318−1335. (7) Yang, Y.; Liu, F.; Liu, X.; Xing, B. NIR Light Controlled Photorelease of siRNA and Its Targeted Intracellular Delivery Based on Upconversion Nanoparticles. Nanoscale 2013, 5, 231−238. (8) Zhang, L.; Yin, Q.-H.; Li, J.-M.; Huang, H.-Y.; Wu, Q.; Mao, Z.W. Functionalization of Dendritic Polyethylene with Cationic Poly(pphenylene ethynylene) Enables Efficient siRNA Delivery for Gene Silencing. J. Mater. Chem. B 2013, 1, 2245−2251. (9) Jiang, R.; Lu, X.; Yang, M.; Deng, W.; Fan, Q.; Huang, W. Monodispersed Brush-like Conjugated Polyelectrolyte Nanoparticles with Efficient and Visualized siRNA Delivery for Gene Silencing. Biomacromolecules 2013, 14, 3643−3652. (10) Kim, I.-B.; Phillips, R.; Bunz, U. H. F. Carboxylate Group SideChain Density Modulates the pH-Dependent Optical Properties of PPEs. Macromolecules 2007, 40, 5290−5293. (11) Fan, Q.-L.; Lu, S.; Lai, Y.-H.; Hou, X.-Y.; Huang, W. Synthesis, Characterization, and Fluorescence Quenching of Novel Cationic Phenyl-Substituted Poly(p-phenylenevinylene)s. Macromolecules 2003, 36, 6976−6984. (12) McQuade, D. T.; Hegedus, A. H.; Swager, T. M. Signal Amplification of a “Turn-On” Sensor: Harvesting the Light Captured by a Conjugated Polymer. J. Am. Chem. Soc. 2000, 122, 12389− 12390. (13) Liu, X.; Xiang, J.; Zhu, D.; Jiang, L.; Zhou, Z.; Tang, J.; Liu, X.; Huang, Y.; Shen, Y. Fusogenic Reactive Oxygen Species Triggered Charge-Reversal Vector for Effective Gene Delivery. Adv. Mater. 2016, 28, 1743−1752. (14) Naito, M.; Ishii, T.; Matsumoto, A.; Miyata, K.; Miyahara, Y.; Kataoka, K. A Phenylboronate-functionalized Polyion Complex Micelle for ATP-triggered Release of siRNA. Angew. Chem., Int. Ed. 2012, 51, 10751−10755. (15) Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M. A Review of Stimuli-responsive Nanocarriers for Drug and Gene Delivery. J. Controlled Release 2008, 126, 187−204. (16) van Vliet, A. H. M.; Kuipers, E. J.; Stoof, J.; Poppelaars, S. W.; Kusters, J. G. Acid-responsive Gene Induction of Ammonia-producing Enzymes in Helicobacter pylori is Mediated via a Metal-responsive Repressor Cascade. Infect. Immun. 2004, 72, 766−773. (17) Viger, M. L.; Grossman, M.; Fomina, N.; Almutairi, A. Low Power Upconverted Near-IR Light for Efficient Polymeric Nanoparticle Degradation and Cargo Release. Adv. Mater. 2013, 25, 3733− 3738. (18) Qi, J.; Chen, C.; Zhang, X.; Hu, X.; Ji, S.; Kwok, R. T. K.; Lam, J. W. Y.; Ding, D.; Tang, B. Z. Light-Driven Transformable Optical Agent with Adaptive Functions for Boosting Cancer Surgery Outcomes. Nat. Commun. 2018, 9, 1848−1860.

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CONCLUSION In summary, this study demonstrates that PPENBr-ONB can be combined with siRNA for two-photon induced gene therapy with promoted gene release. PPENBr-ONB in response to 720 nm irradiation can accelerate siRNA release induced by charge changes after photodegradation of ONB for gene therapy. Such a simple and useful platform has shown high-effective gene therapy. Thus, this siRNA delivery system based on two-photon-triggered PPENBr-ONB holds great prospects for the development of gene therapy and therefore can be exploited as a platform for nucleic acid-based therapeutics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00059. Synthesis of PPENBr-ONB and their photolytic process; 1 H NMR and 13C NMR of 4,5-dimethoxy-2-nitrobenzyl 2-bromoacetate; 1H NMR of PPEN and PPENBr-ONB; FTIR of PPEN and PPENBr-ONB; stability of PPENBrONB/siRNA complexes; two-photon fluorescence spectra of PPEN, PPENBr-ONB, and PPENBr-ONB after 720 nm irradiation; in vitro cell viability of PPENBrONB and PPENBr-ONB/siRNA complex (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wenbo Hu: 0000-0001-5233-8183 Quli Fan: 0000-0002-9387-0165 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 21674048, 21574064, and 61805118), the Natural Science Foundation of Jiangsu Province of China (No. BK20171020), the China Postdoctoral Science Foundation (Nos. 2017M621733 and 2018T110488), 333 project of Jiangsu province (No. BRA2016379), Shenzhen Basic Research Project of Science and Technology under Grant No. JCYJ20170302142433007, open research fund of Key Laboratory for Organic Electronics, I

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Article

ACS Applied Bio Materials (19) Ni, X.; Zhang, X.; Duan, X.; Zheng, H.-L.; Xue, X.-S.; Ding, D. Near-Infrared Afterglow Luminescent Aggregation-Induced Emission Dots with Ultrahigh Tumor-to-Liver Signal Ratio for Promoted Image-Guided Cancer Surgery. Nano Lett. 2019, 19, 318−330. (20) Gao, H.; Zhang, X.; Chen, C.; Li, K.; Ding, D. Unity Makes Strength: How Aggregation-Induced Emission Luminogens Advance the Biomedical Field. Adv. Biosyst. 2018, 2, 1800074. (21) Sobolciak, P.; Spirek, M.; Katrlik, J.; Gemeiner, P.; Lacik, I.; Kasak, P. Light-Switchable Polymer from Cationic to Zwitterionic Form: Synthesis, Characterization, and Interactions with DNA and Bacterial Cells. Macromol. Rapid Commun. 2013, 34, 635−639. (22) Jayakumar, M. K.; Idris, N. M.; Zhang, Y. Remote Activation of Biomolecules in Deep Tissues Using Near-Infrared-to-UV Upconversion Nanotransducers. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 8483− 8488. (23) Ji, W.; Li, N.; Chen, D.; Qi, X.; Sha, W.; Jiao, Y.; Xu, Q.; Lu, J. Coumarin-containing Photo-Responsive Nanocomposites for NIR Light-Triggered Controlled Drug Release via a Two-photon Process. J. Mater. Chem. B 2013, 1, 5942−5949. (24) Sun, L.; Yang, Y.; Dong, C. M.; Wei, Y. Two-Photon-Sensitive and Sugar-Targeted Nanocarriers From Degradable and Dendritic Amphiphiles. Small 2011, 7, 401−406. (25) Croissant, J.; Maynadier, M.; Gallud, A.; Peindy N’dongo, H.; Nyalosaso, J. L.; Derrien, G.; Charnay, C.; Durand, J. O.; Raehm, L.; Serein-Spirau, F.; Cheminet, N.; Jarrosson, T.; Mongin, O.; Blanchard-Desce, M.; Gary-Bobo, M.; Garcia, M.; Lu, J.; Tamanoi, F.; Tarn, D.; Guardado-Alvarez, T. M.; Zink, J. I. Two-PhotonTriggered Drug Delivery in Cancer Cells Using Nanoimpellers. Angew. Chem., Int. Ed. 2013, 52, 13813−13817. (26) Zhao, H.; Hou, B.; Tang, Y.; Hu, W.; Yin, C.; Ji, Y.; Lu, X.; Fan, Q.; Huang, W. O-Nitrobenzyl-alt-(phenylethynyl)benzene Copolymer-Based Nanoaggregates with Highly Efficient Two-PhotonTriggered Degradable Properties via a FRET Process. Polym. Chem. 2016, 7, 3117−3125. (27) Zhang, Z.; Lu, X.; Fan, Q.; Hu, W.; Huang, W. Conjugated Polyelectrolyte Brushes with Extremely High Charge Density for Improved Energy Transfer and Fluorescence Quenching Applications. Polym. Chem. 2011, 2, 2369−2377. (28) Parker, C. A.; Rees, W. T. Correction of Fluorescence Spectra and Measurement of Fluorescence Quantum Efficiency. Analyst 1960, 85, 587−600. (29) Xu, C.; Webb, W. W. Measurement of Two-Photon Excitation Cross Sections of Molecular Fluorophores with Data from 690 to 1050 nm. J. Opt. Soc. Am. B 1996, 13, 481−491. (30) Makarov, N. S.; Drobizhev, M.; Rebane, A. Two-Photon Absorption Standards in the 550−1600 Nm Excitation Wavelength Range. Opt. Express 2008, 16, 4029−4047. (31) Magde, D.; Wong, R.; Seybold, P. G. Fluorescence Quantum Yields and Their Relation to Lifetimes of Rhodamine 6g and Fluorescein in Nine Solvents: Improved Absolute Standards for Quantum Yields. Photochem. Photobiol. 2002, 75, 327−334. (32) Zhao, H.; Hu, W.; Ma, H.; Jiang, R.; Tang, Y.; Ji, Y.; Lu, X.; Hou, B.; Deng, W.; Huang, W.; Fan, Q. Photo-Induced ChargeVariable Conjugated Polyelectrolyte Brushes Encapsulating Upconversion Nanoparticles for Promoted siRNA Release and Collaborative Photodynamic Therapy under NIR Light Irradiation. Adv. Funct. Mater. 2017, 27, 1702592. (33) Pu, K.-Y.; Chen, Y.; Qi, X.-Y.; Qin, C.-Y.; Chen, Q.-Q.; Wang, H.-Y.; Deng, Y.; Fan, Q.-L.; Huang, Y.-Q.; Liu, S.-J.; Wei, W.; Peng, B.; Huang, W. Synthesis of Grafted Poly(p-phenyleneethynylene) with Energy Donor−Acceptor Architecture via Atom Transfer Radical Polymerization: Towards Nonaggregating and Hole-Facilitating Light-Emitting Material. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 3776−3787. (34) Chen, R.; Ta, V. D.; Xiao, F.; Zhang, Q.; Sun, H. Multicolor Hybrid Upconversion Nanoparticles and Their Improved Performance as Luminescence Temperature Sensors Due to Energy Transfer. Small 2013, 9, 1052−1057.

(35) Harpur, A. G.; Wouters, F. S.; Bastiaens, P. I. Imaging FRET Between Spectrally Similar GFP Molecules in Single Cells. Nat. Biotechnol. 2001, 19, 167−169. (36) Lutich, A. A.; Jiang, G.; Susha, A. S.; Rogach, A. L.; Stefani, F. D.; Feldmann, J. Energy Transfer Versus Charge Separation in TypeII Hybrid Organic-Inorganic Nanocomposites. Nano Lett. 2009, 9, 2636−2640. (37) Cucinotta, F.; Carniato, F.; Devaux, A.; De Cola, L.; Marchese, L. Efficient Photoinduced Energy Transfer in a Newly Developed Hybrid SBA-15 Photonic Antenna. Chem. - Eur. J. 2012, 18, 15310− 15315. (38) Pinto, M. R.; Kristal, B. M.; Schanze, K. S. A Water-soluble Poly(phenylene ethynylene) with Pendant Phosphonate Groups. Synthesis, Photophysics, and Layer-By-Layer Self-Assembled Films. Langmuir 2003, 19, 6523−6533. (39) Fan, Q.-L.; Zhou, Y.; Lu, X.-M.; Hou, X.-Y.; Huang, W. WaterSoluble Cationic Poly(p-phenyleneethynylene)s (PPEs): Effects of Acidity and Ionic Strength on Optical Behavior. Macromolecules 2005, 38, 2927−2936. (40) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo. Science 2003, 300, 1434−6. (41) Strebhardt, K.; Ullrich, A. Targeting Polo-like Kinase 1 for Cancer Therapy. Nat. Rev. Cancer 2006, 6, 321−330.

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