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Mar 7, 2017 - ABSTRACT: The stimuli-responsive polymeric nanocarriers have been studied extensively, and their structural changes in cells are importa...
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In Situ Monitoring Intracellular Structural Change of Nanovehicles through Photoacoustic Signals Based on Phenylboronate-Linked RGD-Dextran/Purpurin 18 Conjugates Dong-Bing Cheng,† Guo-Bin Qi,† Jing-Qi Wang,† Yong Cong,† Fu-Hua Liu,† Haijun Yu,‡ Zeng-Ying Qiao,*,† and Hao Wang*,† †

CAS Center for Excellence in Nanoscience, Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing, 100190, China ‡ State Key Laboratory of Drug Research and Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China S Supporting Information *

ABSTRACT: The stimuli-responsive polymeric nanocarriers have been studied extensively, and their structural changes in cells are important for the controlled intracellular drug release. The present work reported RGD-dextran/purpurin 18 conjugates with pH-responsive phenylboronate as spacer for monitoring the structural change of nanovehicles through ratiometric photoacoustic (PA) signal. Phenylboronic acid modified purpurin 18 (NPBA-P18) could attach onto the RGD-decorated dextran (RGD-Dex), and the resulting RGD-Dex/NPBA-P18 (RDNP) conjugates with different molar ratios of RGD-Dex and NPBA-P18 were prepared. When the moles of NPBA-P18 were equivalent to more than triple of RGD-Dex, the single-stranded RDNP conjugates could self-assemble into nanoparticles in aqueous solution due to the fairly strong hydrophobicity of NPBA-P18. The pHresponsive aggregations of NPBA-P18 were investigated by UV−vis, fluorescence, and circular dichroism spectra, as well as transmission electron microscope. Based on distinct PA signals between monomeric and aggregated state, ratiometric PA signal of I750/I710 could be presented to trace the structural change progress. Compared with RDNP single chains, the nanoparticles exhibited effective cellular internalization through endocytosis pathway. Furthermore, the nanoparticles could form well-ordered aggregates responding to intracellular acidic environment, and the resulting structural change was also monitored by ratiometric PA signal. Therefore, the noninvasive PA approach could provide a deep insight into monitoring the intracellular structural change process of stimuli-responsive nanocarriers.

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INTRODUCTION Over the past several years, the pathology-specific responsive nanovehicles have emerged as the powerful vehicles for improving the therapeutic efficacy and diminishing adverse effects in cancer therapy.1−5 The nanovehicles could protect the hydrophobic therapeutic agents from the aqueous environment, prolong circulation time, and accumulate at the tumor sites through targeting effect. The responsive properties enable the controlled release of therapeutic agents responding to specific stimuli associated with tumor microenvironment. Considering the pH values of intracellular compartments, such as the endosome/lysosome organelles (pH 5.0−6.0), are lower than that of blood circulation (pH 7.4), many researches have focused on the pH-responsive nanovehicles for realizing the fast release of therapeutic agents in targeted cells.6−9 pH-responsive nanovehicles mostly consist of acid-sensitive bonds, such as hydrazone, acetal, orthoester, and amide, which © XXXX American Chemical Society

could induce the structural change of nanovehicles in response to the low pH value of lysosome.10−12 Among the acid-sensitive bonds, the covalent phenylboronate bond has been developed widely due to the unique pH-reversible characteristic, which can spontaneously form at neutral condition and is susceptible to be hydrolyzed under acidic conditions.13−16 Zhang et al. reported the formation of pH-responsive boronate-linked dextran/cholesterol nanoparticles for improving drug efficacy. The prepared nanoparticles showed sufficient structural stability under physiological conditions while being dissociated in the simulated acid environment, resulting in the accelerated release of loaded doxorubicin (DOX) in tumor cells and improved antitumor chemotherapy.17 Lam et al. reported Received: December 26, 2016 Revised: March 3, 2017 Published: March 7, 2017 A

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Biomacromolecules Scheme 1. Illustration of pH-Responsive RDNP Conjugates for Intracellular Acid Modulated Ratiometric PA Signala

a

(A) Structural formula of RDNP conjugates. (B) State of RDNP conjugates in aqueous solution and their acid-triggered aggregation. (C) Ratiometric PA signal of different aggregation states of NPBA.

particles with cytotoxic peptide KLAK could disrupt the eukaryotic mitochondrion, and the overgenarated ROS associated with disrupted mitochondrial membranes could simulate the swelling of nanoparticles, which resulted in the release of SQ and subsequent increase of PA signal with Haggregates of SQ.26 Dextran derived from the natural materials has been applied as drug nanocarriers owing to its good biocompatibility.27−29 Herein, a pH-responsive PA system based on biocompatible peptide RGD decorated dextran (RGD-Dex) and phenylboronic acid modified P18 (NPBA-P18) was constructed for monitoring intracellular acid-induced structural change process of nanovehicles (Scheme 1). The RGD-Dex/NPBA-P18 (RDNP) conjugates with different molar ratios of RGD-Dex and NPBA-P18 were prepared by controlling their feed ratios. Due to the hydrophobicity of P18, the RDNP conjugates could self-assemble into nanoparticles, while the moles of NPBA-P18 were equivalent to more than triple of RGD-Dex in aqueous solution. Both the monomeric and nanogranulated state of NPBA-P18 could aggregate into nanofibers in response to acidic environment, resulting in the change of PA signals. Meanwhile, the dynamic equilibrium of these states along with pH-reversible phenylboronate linkage was monitored and studied by ratiometric PA signal. As the vehicles, the nanoparticles with tumor-targeting peptide could be effectively taken up by human cervical cancer HeLa cells through cellular endocytosis, and the structural change process of nanoparticles was successfully imaged according to the ratiometric PA signal. This type of nanoparticles conjugated with PA molecule paves the way for monitoring the intracellular structural change process through noninvasive approach, exhibiting the great potential as the self-reporting nanovehicles in controlled release of therapeutic agents in living cells.

boronate cross-linked PEG-cholic acid nanocarriers for drug delivery. The release of paclitaxel from the boronate crosslinked micelles was significantly slower than that from noncross-linked micelles at pH 7.4, but could be accelerated by the acidic pH values.18 Although most of the reports have focused on the release of cargos from nanovehicles, it is still unknown how the structure of nanovehicles changes in living targeted cells. The attributes of nanovehicle structural stability in physiological environment and intracellular acid triggered dissociation are important for controlled release of therapeutic agents, and thus, the in situ monitoring of the structural change of nanovehicles in cells could provide the intuitional and deeper insight for construction of pH-responsive drug delivery systems. Photoacoustic tomography (PAT, also called optoacoustic tomography) as a noninvasive laser-induced technique has been widely applied for imaging of organelles, cells, tissues, and organs.19−21 Compared with the conventional semiquantitative fluorescence method, the PAT exhibited deeper tissue penetration ability and higher spatial resolution, which could be more accurate and efficient in bioimaging. As the photosensitizer, purpurin 18 (P18) could aggregate into wellordered supramolecular complex due to strong intermolecular cofacial π−π stacking interactions in aqueous solution, which exhibited distinct photoacoustic (PA) signals between monomeric and well-ordered aggregated state.22,23 Our group has reported the small-molecule P18/peptide conjugates, responding to special microenvironment and self-assembling into supramolecular structure in vivo, which have successfully been applied for tumor and infection imaging.24 Meanwhile, our group also developed the PAT imaging method to evaluate the drug release profiles from polymeric carriers. The loaded drug DOX and near-infrared (NIR) dye squaraine (SQ) could both be released in acidic environment, and the PA signal intensity changed with the aggregation of released SQ.25 Recently, SQ was loaded in reactive oxygen species (ROS) sensitive polymer−peptide therapeutic nanoparticles for evaluating the antitumor performance by in situ PA imaging. The nano-

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EXPERIMENTAL SECTION

Materials. Dextran (Dex, Mn = 5000), 3-(aminomethyl) phenylboronic acid hydrochloride (NPBA·HCl) and acryloyl chloride were purchased from Sima-Aldrich Chemical Co., Ltd. P18 was obtained B

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Biomacromolecules Scheme 2. Synthesis Route of RDNPa

(A) Synthesis of NPBA-P18 (EDC·HCl, DMAP, DMSO, 25 °C, 3 h). (B) Synthesis of acryl-Dex (Et3N, DMF, 25 °C, 24 h) and RGD-Dex (Et3N, DMSO, 25 °C, 24 h). (C) Synthesis of RDNP conjugates (DMSO, 25 °C, 8 h).

a

Synthesis of NPBA-P18. The NPBA-P18 was synthesized according the previously reported method.30 Briefly, P18 (100 mg, 0.18 mmol), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 36 mg, 0.20 mmol), and 4-dimethylaminopyridine (DMAP, 24 mg, 0.2 mmol) was dissolved in 10 mL of DMSO, and then NPBA·HCl (113 mg, 0.60 mmol) was added under stirring. The reaction process was monitored by thin-layer chromatography (TLC). After 3 h, the resultant solution was poured into CH2Cl2 and washed with saline solution. The organic layer was dried over MgSO4 and the solvent was removed by rotary evaporation, the residue was precipitated into cold diethyl ether/ethyl acetate (1:1) mixture and dried under vacuum to obtain the amaranthine solid NPBA-P18. Synthesis of RDNP Conjugates. NPBA-P18 (5 mg, 7.29 μmol) and various amounts of RGD-Dex (52 mg, 7.29 μmol; 26 mg, 3.65 μmol; 17 mg 2.43 μmol; 13 mg, 1.82 μmol; 10 mg, 1.46 μmol) was dissolved in anhydrous DMSO (5 mL), and then 4 Å molecular sieves (1 g) were added to the solution and the reaction was stirred for 8 h at room temperature. The reaction mixture was dialyzed (MWCO: 2000 Da) against distilled water for 24 h, and the resulting solution was then lyophilized to obtain purple solid RDNP. Preparation of Nanoparticles. RDNP nanoparticles were prepared by dialysis method. The RDNP conjugates were dissolved in 2 mL of DMSO under stirring, and then 4 mL of phosphate buffered solution (PB, 0.01 M, pH 7.4) was added dropwise. The solution was dialyzed (MWCO: 2000 Da) against PB for 24 h to form the RDNP nanoparticles dispersion. Determination of Critical Aggregation Concentration (CAC). The CAC of RDNP conjugates was determined using pyrene as a fluorescent probe. Briefly, 50 μL of pyrene solution in acetone (4.8 × 10−4 M) was added to a 5 mL centrifuge tube, and then acetone was evaporated completely. A series of RDNP solutions with different concentrations were added to the tube, while the concentration of pyrene was fixed at 6 × 10−6 M. The excitation spectra were recorded at an emission wavelength of 393 nm, and the intensity ratio (I338/I335) was analyzed as a function of the RDNP concentration. The CAC was

from Shanghai Xianhui Pharmaceutical Co., Ltd. CGGRGD peptides were prepared using standard Fmoc solid-phase peptide synthesis method and purified by reverse-phase high-performance liquid chromatography. Mito-Tracker Green and cell counting kit-8 (CCK8) were obtained from Beyotime institute of Biotechnology. LysoTracker Green DND-26, 3-Dulbecco’s Modified Eagle’s Medium (DMEM), and fetal bovine serum (FBS) were purchased from Invitrogen. The HeLa cell line was purchased from the cell culture center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Other solvents and reagents were of analytical grade and purified by general methods. Characterization. 1H NMR spectra were recorded on a Bruker ARX 400 MHz spectrometer using DMSO-d6 or D2O as solvents. UV−vis spectra were recorded on a Shimadzu UV-2600 spectrometer. Fluorescence spectra were recorded by F-280 spectrofluorometer. CD spectra were obtained on a circular dichroism spectrometer (JASCO J 1500, Japan). Transmission electron microscope (TEM) observations were conducted on a Tecnai G2 20 S-TWIN electron microscope. Nano-ZS 3600 (Malvern Instruments, U.K.) was used to measure the size distribution of the nanoparticle dispersion. An average value was determined by three repeated measurements for each sample. Synthesis of RGD-Dex. The RGD-Dex was synthesized in two steps. First, the Dex (1 g, 0.20 mmol) and triethylamine (Et3N, 50 μL, 0.36 mmol) were dissolved in 20 mL of N,N-dimethyformamide (DMF). After cooling at 0 °C, acryloyl chloride (8 μL, 0.10 mmol) in 8 mL of dichloromethane solution was added dropwise to the stirred solution. The reaction proceeded for 24 h at room temperature and then dialyzed (MWCO: 1000 Da) for 48 h against distilled water. The white solid (named acryl-Dex) was obtained after lyophilization. Then, acryl-Dex (518 mg, 0.10 mmol) and CGGRGD (100 mg, 0.18 mmol) was dissolved in 5 mL of dimethyl sulfoxide (DMSO), and Et3N (10 μL, 0.07 mmol) was added at room temperature. The reaction was carried out for 24 h and dialyzed (MWCO: 1000 Da) for 48 h against distilled water, the polymer RGD-Dex finally was obtained as a white solid after lyophilization. C

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Figure 1. 1H NMR (400 MHz, DMSO-d6) spectra of (A) NPBA-P18 (1.0 mg mL−1) and (B) acryl-Dex (1.0 mg mL−1).

Figure 2. 1H NMR (400 MHz, DMSO-d6) spectra of (A) RGD-Dex (1.0 mg mL−1) and (B) RDNP conjugates (2.0 mg mL−1). were recorded through mean pixel intensity in the images. The PA signal of NPBA-P18 in DMSO or water was also monitored as the control. Cytotoxicity Assay in HeLa Cells. The cytotoxicity assay was performed with HeLa cells by CCK-8 assay. HeLa cells were seeded in a 96-well culture plate (6000 cells/well) with 100 μL of DMEM containing 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5%

determined based on the intersection point at low concentration on the plot. PA Signal Detection. PA signal was recorded at 37 °C using a MSOT 128 multispectral optoacoustic tomography. The RDNP solutions were added into the agarose tube with the same concentration of NPBA-P18, and then the PA signal was detected under excitation wavelength at a range of 680−770 nm and the data D

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Table 1. Properties of RDNP Conjugates (Mean ± S.D., n = 3)

CO2 atmosphere for 24 h, and the cells were exposed to RDNP at a series of concentrations for another 24 h. Then, 10 μL of CCK-8 solutions was added to each well and further incubated for 2 h. The concentration of the proliferating cells in each well was confirmed using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm, respectively. The relative cell viability was calculated according to the following equation:

RGD-Dex:NPBA-P18 polymer

feed ratio

measured ratioa

P1 P2 P3 P4 P5

1:1 1:2 1:3 1:4 1:5

1:0.94 1:1.86 1:2.77 1:3.79 1:4.13

cell viability(%) = (A samples − A 0)/(Acontrol − A 0) × 100% where Asamples was obtained in the presence of nanoparticles dispersions and A0 was obtained with complete DMEM. All the experiments were performed in triplicate. Intracellular Uptake. HeLa cells was cultured at a density of 1 × 105 cells in a confocal microscope dish in DMEM media at 37 °C and a humidified atmosphere containing 5% CO2 for 24 h. The RDNP solutions (with 10 μM RGD concentration) were added to each well and further incubated for 2 or 3 h. The medium was removed and the cells were washed three times with PBS. Then the lysosomes were stained with Lyso-Tracker Green DND-26 (500 nM) at 37 °C for 30 min. Finally, the cells were washed three times again with PBS and incubated with 1 mL of DMEM. The fluorescence in cells was observed with a Zeiss LSM710 confocal laser scanning microscope under an oil 63× objective lens. PA Signal Detection in HeLa Cells. HeLa cells with a density of 1 × 106 were seeded in a 10 cm dish and incubated for 24 h, and then the RDNP solutions (with 100 μM NPBA-P18 concentration) were added to each well and further incubated. After a preset time, the medium was removed and the cells were washed three times with PBS. Subsequently, the cells were harvested by trypsin and mixed with 1% ultrapure agarose (60 °C), which were added to the agarose gel tube prepared beforehand. The PA signal was detected under excitation wavelength at a range of 680−770 nm and the data were recorded through mean pixel intensity in the images.

CAC (μg mL−1)

diameterb (nm)

PDIc

36.2 8.9 2.9

11.2 ± 4.7 12.5 ± 3.9 355.5 ± 10.7 128.7 ± 5.3 83.3 ± 5.3

0.255 0.272 0.237 0.163 0.189

a

Measured by UV−vis spectrometer at 700 nm. bHydrodynamic diameter of RDNP conjugates measured by DLS in PB (0.01 M, pH 7.4). cPolydispersity index of RDNP conjugates obtained from DLS measurement.

UV−vis absorbance at 700 nm, which agreed well with their feed ratios. The P5 with obviously decreased moles of NPBAP18 could be possibly attributed to the critical state of watersolubility of RDNP nanoparticles. Aggregation Properties of RDNP Conjugates. The CAC was used to determine the nanoparticle-forming ability of RDNP conjugates. The CAC values were calculated from the plot of the excitation intensity ratio I338/I335 as a function of the concentration of the RDNP conjugates. As shown in Figure 3A,

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RESULTS AND DISCUSSION Synthesis of RDNP Conjugates. The RDNP conjugates with pH-responsive phenylboronate linkages were developed for monitoring the intracellular structural change process with PAT approach (Scheme 2). The structure of NPBA-P18 was confirmed by 1H NMR spectrum shown in Figure 1A. The peaks at 5.3, 6.7, 7.2, 7.9, and 9.2 ppm were assigned to the functional NPBA moiety, while other proton signals were assigned to P18 moieties. The appearance of peak at 9.5 ppm from amide proton signal (peak f) suggested the linkage of NPBA and P18. The RGD-Dex was obtained by modifying hydroxyl group of Dex with acryloyl chloride followed by Michael addition reaction with an excess of tumor-targeting peptides CGGRGD in organic solvent. 1H NMR spectrum of acryl-Dex was presented in Figure 1B, and the characteristic peaks of acrylate groups were observed at 5.8−6.4 ppm. The ∼11% (molar ratio) of glucose units were modified by acrylate groups, which was calculated by the integral ratio of Dex signals at 4.7 ppm and acrylate signals. After Michael-addition reaction between acrylate groups of acryl-Dex and thiol groups of CGGRGD (Figure S1), the acrylate signals completely disappeared and the RGD signals were clearly shown (Figure 2A), which demonstrated the successful synthesis of RGD-Dex. Phenylboronic acid could covalently bond with the diol group of Dex, and the structure of RDNP conjugates was confirmed by 1H NMR.31,32 As shown in Figure 2B, the signals of phenyl groups were clearly shown. The RDNP with different feed molar ratios of RGD-Dex and NPBA-P18 (1:1, 1:2, 1:3, 1:4, and 1:5) were denoted as P1, P2, P3, P4, and P5, respectively (Table 1). Based on the standard calibration curve of NPBA-P18 at 700 nm in DMSO (Figure S2), the molar compositions of P1, P2, P3, and P4 were calculated from the

Figure 3. (A) CAC values determined by I338/I335 ratio from pyrene as a function of concentration of RDNP conjugates. (B) Particle size of RDNP conjugates in PB (0.1 mg mL−1, 0.01 M, pH 7.4) measured by DLS.

the CAC values of P1 and P2 were higher than 500 mg L−1, which demonstrated the P1 and P2 could not form nanoparticles in aqueous solution at the experimental concentration used in this study. The CAC values of P3, P4, and P5 were 36.2, 8.9, and 2.9 mg L−1, respectively, suggesting that the large fraction of hydrophobic P18 could induce RDNP conjugates to form the core−shell nanoparticles. The CAC values decreased with the increased P18 fraction in RDNP conjugates due to the E

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Figure 4. TEM images of RDNP conjugates in PB (0.01 M, pH 7.4) or AB (0.01 M, pH 5.0) after 4 h. The concentration of RDNP was 0.1 mg mL−1.

enhanced hydrophobicity.33,34 Therefore, RDNPs with 0.1 mg mL−1 were used for further study to ensure the aggregation of P3−P5 and single chain state of P1−P2. The average hydrodynamic diameters of RDNPs (0.1 mg mL−1) measured by DLS were consistent with CAC values (Figure 3B). The particle sizes of P1 and P2 were about 10 nm, which was similar to the Dex (Figure S3), proving the singlestranded state of P1 and P2. The P3, P4, and P5 exhibited obviously larger sizes, implying the formation of nanoparticles. The corresponding particle sizes became smaller with the increased moles of hydrophobic NPBA-P18, which could be attributed to their enhanced interaction. Therefore, P5 showed more compact nanostructure with smaller size. TEM observation (Figure 4) showed the P3, P4, and P5 dispersed in spherical shape with average sizes of 46 ± 4, 30 ± 3, and 23 ± 3 nm, respectively. Due to the possible dehydration of the polymer chains in dry state, the average diameters of copolymeric micelles on the TEM image were smaller than that measured by DLS.35 Furthermore, the variation trends of P1−P5 particle size measured by TEM were in good agreement with the DLS results. The aggregation behaviors of nanoparticles were further confirmed by 1H NMR spectroscopy in D2O (Figure S4). For P1 and P2, the proton signals of both RGD-Dex and NPBAP18 could be observed in D2O. While for P3, P4, and P5, the proton signals of NPBA-P18 disappeared, which could be attributed to the limited molecular motion of aggregated NPBA-P18. Therefore, we concluded that P3−P5 selfassembled into core−shell nanoparticles with NPBA-P18 as the hydrophobic core and Dex as the hydrophilic shell to stabilize the nanoparticles.17,36,37 However, P1 and P2 could dissolve into the aqueous solution with single chain state, exhibiting no obvious aggregation. Aggregation of P18 in RDNP Conjugates. P18 exhibited the unique PA signal changes in monomeric and well-ordered aggregated state, which could monitor structural change of RDNP conjugates. The aggregation of NPBA-P18 was first investigated by UV−vis and fluorescence analysis (Figure 5A,B). With the decrease of DMSO in aqueous solution, the Qy bands appeared a red-shift (from 700 to 724 nm) and became broadened, accompanied by fluorescence quenching. These phenomena were in agreement with previous reports,

Figure 5. UV−vis (A) and fluorescence spectra (B) of NPBA-P18 in different mixture solutions of DMSO/H2O (100%−1%). (C) CD spectra of NPBA-P18 as monomers and nanofibers. The final concentration of NPBA-P18 was 10 μM.

F

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Figure 6. UV−vis (A), fluorescence (B), and CD (C) spectra of RDNP conjugates in PB (0.01 M, pH 7.4) or AB (0.01 M, pH 5.0) after 4 h. (D) The ratiometric photoacoustic signals of RDNP conjugates in PB (0.01 M, pH 7.4). The final concentration of NPBA-P18 was 10 μM.

in accordance with the results of UV−vis and fluorescence spectra. pH-Modulated Aggregation of RDNP Conjugates. Because of the acid-induced cleavage characteristic of phenylboronate linkage, the RDNP conjugates could be responsive to intracellular acid, and then the hydrolyzed NPBA-P18 gradually aggregated into well-ordered nanofibers.44,45 As shown in Figure 6A,B, the UV−vis and fluorescence spectra of P5 at pH 5.0 were similar to the NPBA-P18 in aqueous solution. More obvious red-shifted (from 700 to 725 nm), broadened Qy band and declined fluorescence were observed at pH 5.0 than that at pH 7.4. Likewise, the other RDNP conjugates exhibited the similar UV−vis and fluorescence spectra at pH 5.0 (Figure S6). Therefore, NPBA was cleaved from RDNP conjugates in acidic environment, and subsequently the unbonded NPBA-P18 could reassemble into more aggregates through hydrophobic and π−π interactions. In addition, the morphology, UV−vis, and fluorescence spectra of nanoparticles kept same when the pH value of nanoparticle solution was restored from 5.0 to 7.4, which demonstrated the NPBA-P18 aggregates could be stable in neutral environment (Figure S7). The pH-responsive aggregation of P18 was also confirmed by CD spectra (Figure 6C). At pH 5.0, P5 showed similar bisignate signals with the aggregated state of NPBA-P18, while the bisignate signals were enhanced obviously in Qy band area, which suggested the unbonded NPBA-P18 could form the well−ordered supramolecular aggregations. Likewise, the other RDNP conjugates exhibited the similar CD spectra at pH 5.0 (Figure S9). The TEM observation also showed the RDNP conjugates dispersed with nanofiber morphologies at pH 5.0, which further demonstrated the structure of RDNP conjugates could be destroyed in response to the reduced pH value and the divorced NPBA-P18 could form the supramolecular aggregations in nanofiber morphology (Figure 4). All the above results revealed that the structure of RDNP conjugates could change along with the cleavage of phenylboronate and

suggesting the NPBA-P18 formed aggregates through hydrophobic and π−π interactions in aqueous solution.24,38 After NPBA-P18 was linked onto RGD-Dex main chains, the P18 aggregation behaviors in RDNP conjugates were studied. As shown in Figure 6A,B, the single-stranded state of P1 and P2 showed the similar UV−vis and fluorescence spectra with NPBA-P18 in DMSO, indicating the monomeric P18 existed in P1 and P2 chains. While the intensities of fluorescence of nanogranulated state (P3, P4, and P5) were considerably attenuated, and the Qy bands slightly broadened and redshifted (from 700 to 714 nm) simultaneously. The state of hydrophobic NPBA-P18 in the core of RDNP nanoparticles might contain the aggregates and monomers, which aroused the apparent change of UV−vis and fluorescence spectra.39 CD spectra also confirmed slight aggregation of NPBA-P18 in P3− P5 nanoparticles (Figure 6C). 40,41 Comparing to the monomeric state, the Qy band area of aggregated state showed enhanced bisignate signals (Figure 5C), which can be explained by excitonic coupling of the transition dipole moments of the intrinsically chiral P18 molecule.41 With the increased moles of NPBA-P18, the bisignate signals of Qy band area in P3, P4, and P5 were gradually enhanced, which demonstrated the more aggregates in the compact nanoparticles. PA signals of RDNP conjugates have the positive correlation with molar extinction coefficient presented in UV−vis spectra (Figure S5).42,43 According to the lowest and highest PA intensity ratio, the suitable wavelengths for analyzing aggregation were 710 and 750 nm, which could be more rational to monitor the aggregation index (Figure 6D). The I750/I710 values of PA signals for P1 and P2 were 0.34 and 0.41, respectively. In contrast, the I750/I710 values of PA signals for P3, P4, and P5 were 0.63, 0.65, and 0.71, respectively, suggesting the I750/I710 values increased with more aggregation of P18. Therefore, ratiometric PA signal could be used to study the P18 aggregation behaviors in RDNP conjugates, which was G

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Figure 7. CLSM images of HeLa cells incubated with P5 and P2 (10 μM) for 2 h, and lysosomes were labeled with LysoTracker Green DND-26 (500 nM, 30 min). Scale bar: 20 μm.

The cellular uptake pathway of P5 was further verified by incubating HeLa cells with various endocytosis inhibitors for 2 h (Figure S8). A total of 2 mM amiloride, 5 mM β-cyclodextrin (β-CD), and 450 mM hypertonic sucrose were used to inhibit macropinocytosis, caveolae-mediated, and clathrin-mediated endocytosis of P5, respectively. Compared with the control without endocytosis inhibitor, similar intensity of red fluoresence was observed inside cells treated with amiloride, while the weaker intensity was showed in cells treated with βCD and hypertonic sucrose. The inhibition study showed that P5 entered HeLa cells by caveolae-mediated and clathrinmediated endocytosis. Furthermore, the weakest intensity upon treatment of hypertonic sucrose exhibited most efficient entrance inhibition of P5 into cells, indicating that the main pathway for P5 nanoparticle uptake was clathrin-mediated endocytosis. Besides, RGD could be specifically recognized by αvβ3-integrins overexpressed on HeLa cells, which had been acted as a target ligand in nanoparticles to improve the selectivity and cellular uptake in previous studies.48 The weakened red fluoresence was observed when P5 was incubated with free RGD, further suggesting the peptide RGD in the RDNP conjugates could enhance the uptake by HeLa cells (Figure 7). pH-Modulated PA Signals of RDNP Conjugates. In order to investigate the pH-responsive NPBA bonds cleavage, PA signals of P5 were also recorded at pH 5.0 and pH 7.4. Comparing to the invariable values of the I750/I710 of PA signals within 4 h at pH 7.4, the ratio values steadily increased in 3 h at pH 5.0, which appeared in a time-dependent manner (Figures 8A and S9). Because the phenylboronate linkage was stable at pH 7.4 and susceptible to be breakable at pH 5.0, the nanoparticles could stay stable at pH 7.4 and be dissociated at pH 5.0. The dissociation of nanoparticles triggered by acid

subsequent P18 aggregation in acidic environment. According to the distinct PA signals of NPBA-P18 between monomeric and well-ordered aggregated state, the state of P18 aggregation could be applied for monitoring the structural change of nanocarriers by PAT. Cellular Uptake. As above-mentioned, most of pHresponsive nanomaterials entered into cells through an endocytosis pathway, and the release of encapsulated cargos depended on the acid microenvironment of endosomes/ lysosomes.46 The cellular internalization ability is a crucial attribute for intracellular cargo release from nanovehicles. The cellular uptake of the RDNP conjugates was monitored by CLSM in HeLa cells for 2 and 3 h through colocalization of RDNP conjugates (labeled with Cy5) and lysosomes stained by LysoTracker Green DND-26 (Figure 7). The LysoTracker Green DND-26 was used to mark the acidic lysosomes organelles of cells, which presented green fluorescence to distinguish from the red fluoresence (Cy5). Due to the two states in aqueous solution of RDNP conjugates, the singlestranded P2 and nanogranulated P5 were chosen for investigating the cellular internalization ability. Compared with P2, the red fluoresence intensity of P5 was obviously stronger, which indicated cellular internalization of nanoparticles was more effective than single chains. The high interfacial density of nanopariticles could be conducive to the cellular uptake, and similar results have been reported by MacEwan et al.47 CLSM images showed the efficient colocalization of P2 and P5 with lysosomes, indicating that RDNP conjugates entered cells through cellular endocytosis and located in endosome/lysosome to ensure the acid-triggered NPBA hydrolysis. When increasing the incubation time to 3 h, the red fluoresence of P5 became stronger due to the fact that more P5 entered and accumulated in cells. H

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Figure 9. Ratiometric photoacoustic signal (A) and PA images of relative intensity (B) of P5 in HeLa cells within 5 h. Results are presented as the mean ± SD in triplicate. Asterisk (*) denoted statistical significance; statistical significance: **p < 0.01.

Figure 8. Ratiometric PA signals (A) and PA images of relative intensity at 750 nm (B) of P5 in PB (0.01 M, pH 7.4) or AB (0.01 M, pH 5.0) within 4 h. Results are presented as the mean ± SD in triplicate. Asterisk (*) denoted statistical significance; statistical significance: ***p < 0.001.

aggregates in acidic lysosomes, which appeared red-shift of PA signal and the ratio of PA intensity became higher. Similar to the PA ratio values at pH 5.0, after 3 h, the I750/I710 of PA signals kept almost stable, implying the nanoparticles were completely dissociated in HeLa cells. We envisioned that the intracellular structural change process of nanoparticles based on phenylboronate linkages could be accurately detected by ratiometric PA signal. It should be noted that the ratiometric PA signal in HeLa cells was not affected by the cellular internalization but could increase with the dissociation of nanoparticles responding to decreased pH value of lysosome. In addition, the P5 exhibited low cytotoxicity at a wide range of concentration (0−500 mg/L; Figure S11). Therefore, the structural change of nanoparticles was successfully monitored and analyzed by the ratiometric PA method in living cells.

could result in the detachment of NPBA and subsequent formation of supramolecular aggregations, which significantly increased I750/I710 values of PA signals. These results were also consistent with the results of UV−vis, fluorescence and CD spectra. After 3 h, the I750/I710 of PA signals exhibited little variation at pH 5.0, which could be taken as the threshold to determine the complete dissociation of nanoparticles. In addition, the relative PA intensity at I750 corresponding to supramolecular aggregation also exhibited pronouncedly stronger signal in 4 h at pH 5.0 (Figure 8B). Intracellular PA Signal Detection. After the effective cellular internalization, the structural change of P5 in cells was monitored by PAT. Considering the acid-modulation of PA signals, the HeLa cells was treated with NH4Cl (50 mM), which was documented to be able to block the acidification of endosome to lysosome.49 The CLSM showed that comparing to the cells without NH4Cl treatment, the apparently weaker green fluorescence of LysoTracker Green DND-26 and similar red fluorescence intensity of Cy5 were observed in cells with NH4Cl treatment, which verified the blockage of endosomal acidification progression had no effect on cellular uptake (Figure S10). After 1 h incubation, the ratio values of I750/I710 in HeLa cells with NH4Cl treatment remained about 0.71, which was similar to that in PB (0.01 M, pH 7.4). Meanwhile, the ratio values of I750/I710 of intracellular PA signals upon treatment of NH4Cl almost constant in 5 h at pH 7.4, indicating the P5 did not generate more aggregates and still dispersed as nanoparticles in cells (Figure 9). Therefore, the structure of nanoparticles could keep stable in the neutral lysosome, which showed unchanged ratiometric PA signal. In contrast, after 1 h incubation, the ratio values of I750/I710 in HeLa cells without NH4Cl treatment exhibited a small increment, and the I750/I710 of intracellular PA signals gradually increased in the following 2 h, which was in accordance with the PA ratio value changes at pH 5.0. The NPBA-P18 could disengage from RDNP conjugates and form well-ordered

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CONCLUSION

The present work developed RDNP conjugates with phenylboronate as linkage, and the structural change process of nanoparticles in living cells was monitored by ratiometric PA signal. The RDNP was able to form nanoparticles while the moles of NPBA-P18 were equivalent to more than triple of RGD-Dex. Comparing to the single-stranded structure, the nanoparticles appeared obviously enhanced cellular internalization via endocytosis pathway, which was a crucial factor for intracellular cargo release of pH-responsive nanovehicles. The RDNP could form well-ordered aggregates responding to intracellular acidic environment, which exhibited different PA signals. The structural change of RDNP nanoparticles in living cells could be recorded and analyzed by ratiometric PA signal stemmed from the dynamic balance of monomers and aggregates. Therefore, the intracellular structural change process of nanoparticles could be monitored through PAT, and the further studies in vivo were currently underway. I

DOI: 10.1021/acs.biomac.6b01922 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

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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/acs.biomac.6b01922. MALDI-TOF and 1H NMR spectra of CGGRGD, standard calibration curve of NPBA-P18 and additional results of DLS, 1H NMR, UV−vis, fluorescence, CD, CLSM, and PA measurement, as well as the cell viability assay (PDF).

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AUTHOR INFORMATION

Corresponding Authors

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

Hao Wang: 0000-0002-1961-0787 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21374026, 21502035, 51573032, and 21674027) and the National Basic Research Program of China (973 Program, 2013CB932701).

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DOI: 10.1021/acs.biomac.6b01922 Biomacromolecules XXXX, XXX, XXX−XXX