Rational Design of Polymeric Nanoparticles with Tailorable

Research Article www.acsami.org

Rational Design of Polymeric Nanoparticles with Tailorable Biomedical Functions for Cancer Therapy Wenhai Lin,†,‡ Wei Zhang,†,‡ Tingting Sun,†,‡ Shi Liu,† Yu Zhu,§ and Zhigang Xie*,† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China § Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun 130024, P. R. China S Supporting Information *

ABSTRACT: Polymeric nanoparticles (NPs) play a key role in nanoscale formulations for bioimaging, cancer treatment, and theranostics. In this work, we designed and synthesized a series of hydrophobic polymers (P1−6) with different pendent groups via one-step multicomponent Passerini reaction. These polymers possessed similar molecular structures and various biomedical functions. Interestingly, they could self-assemble into stable NPs in aqueous media. All formed NPs were redox sensitive because of the existence of disulfide bonds in the backbone. The stability of NPs in aqueous media with or without glutathione was systematically evaluated and compared. The optical performance, including fluorescence resonance energy transfer, was characterized under different conditions for those polymers with fluorescent components. Importantly, all formed NPs showed good cytocompatibility toward HeLa cells and different biological functions, including drug loading and delivery, bioimaging with variable fluorescence, and photodynamic activity, as evidenced by experiments in vitro and in vivo. These results demonstrate the great potential of multicomponent reaction to customize versatile polymeric nanoparticles for biomedical applications. KEYWORDS: multicomponent Passerini reaction, drug delivery, bioimaging, photodynamic therapy, polymer

■

INTRODUCTION It is challenging to develop a simple and universal method for synthesizing desirable and multifunctional polymers and corresponding nanoparticles. Multicomponent reaction (MCR) is a simple and effective approach to combine multiple segments into one molecule, which is a wonderful substitute to multistep complex synthesis.1−8 The Passerini reaction is one of the important and widely used MCRs.9−13 An isocyanide, a carboxylic acid, and an aldehyde can be integrated into one molecule containing amide and ester bond via the Passerini reaction. This reaction possesses several advantages, such as no need of catalysts, mild reaction condition, and atom economy. In 2011, Meier and others brought the Passerini reaction into the field of polymer science.14 Then, the reaction became upand-coming for polymer synthesis and related applications. There are three ways to utilize it in polymer science: synthesizing the monomer for polymerization, postmodifying the polymers, and direct polymerization via the Passerini reaction.15 Meier et al. kept on synthesizing various polymers through this reaction.16 For example, they could accurately control the polymeric sequence through the Passerini reaction.17 Li’s group also has made a great contribution to polymer synthesis via the Passerini reaction, including functional polycaprolactones, sequence-regulated poly(esteramide)s, and photocleavable polymers.18−20 The Passerini © 2017 American Chemical Society

reaction has greatly simplified the reaction steps and provided prodigious potentials for synthesizing various polymers. Relatively, previous works mainly focus on the polymer synthesis and the optimization of reaction parameters, and little attention is drawn on the synthesis of functional polymers and their applications. Furthermore, these polymers cannot form specific structures, especially nanostructures, which greatly limited their further applications. Recently, our group reported on synthesizing functional polymers via the Passerini reaction for various applications, especially in biological applications.21,22 For example, we reported a cyanine-containing polymer via the Passerini reaction and used it for bimodal imaging and photothermal therapy.23 Although great progress has been made for the Passerini reaction, it is still a big challenge to obtain the polymers with particular structures and functions via the Passerini reaction. Nanoparticles (NPs) possess outstanding performance in various fields, such as electronics, catalysis, analysis, and medicine.24−31 The application of NPs in medicine is expected to overcome the defects of traditional drugs, such as hydrophobic nature, systemic toxicity, low bioavailability, and Received: July 23, 2017 Accepted: August 16, 2017 Published: August 16, 2017 29612

DOI: 10.1021/acsami.7b10763 ACS Appl. Mater. Interfaces 2017, 9, 29612−29622

Research Article

ACS Applied Materials & Interfaces Scheme 1. Various Polymers Synthesized via Multicomponent Passerini Reaction

poor target efficiency.32−38 Polymeric NPs (PNPs) are one of the important formulations for therapeutic agents.39−42 Imaging agents, drugs, and target molecules can be conjugated chemically or encapsulated physically into PNPs. To achieve smart drug release, stimuli-responsive molecules or segments should be endowed into PNPs.43−45 Multifunctional nanomedicine applications usually require very complex and multistep reactions for integrating imaging, targeting, and treatment. We wonder whether there are simple methods, especially one-step methods, to combine imaging agents, therapeutic agents, and stimuli-responsive groups into one molecule or polymer, which also can self-assemble into stable NPs in aqueous media. It is multicomponent Passerini reaction that may be a good solution. We have prepared redox- and light-sensitive BDDIPY-dimer via the Passerini reaction, which can self-assemble into nanocapsules for cellular imaging and therapeutic agent delivery.46,47 In this work, we synthesized a series of poly(ester-amide)s (P1−6) with disulfide bonds via one-pot Passerini reaction. Starting from same 1,6-diisocyanohexane and 3,3-dithiodipropionic acid, the versatile polymers were obtained by varying the structure of aldehyde-containing molecules, which could be imaging and therapeutic agents. All obtained polymers could

self-assemble into stable NPs in aqueous solution. The stability, redox sensitivity, and spectroscopic properties of NPs were systemically evaluated. These NPs with good cytocompatibility showed multifunctional bioapplications, including drug loading and delivery, cellular imaging, and PDT.

■

RESULTS AND DISCUSSION

Synthesis and Characterization of NPs. Multicomponent Passerini reaction is an outstanding method to synthesize polymers.48,49 1,6-Diisocyanohexane, 3,3-dithiodipropionic acid, and a molecule containing an aldehyde group were mixed under mild conditions and then the targeted polymers were successfully obtained as shown in Scheme 1. To evaluate the effect of π-conjugated surface on the Passerini reaction and make synthetic polymers multifunctional, o-nitrobenzaldehyde (compound 1), 1-naphthaldehyde (2), 4-(1,2,2-triphenylvinyl)benzaldehyde (TPE-CHO, 3), 7-diethylamino-4-formylcoumarin (COU-CHO, 4), 4,4-difluoro-8-(40-formylphenyl)-1,3,5,7tetramethyl-4-bora-3a,4a-diaza-s-indacene (BDP-CHO, 5), and 5-{2′-(4″-formylphenyl)ethenyl}-4,4-difluoro-8-phenyl-1,3,5,7tetramethyl-4-bora-3a,4a-diaza-s-indacene (REDBDP-CHO, 6) were chosen to synthesize corresponding polymers (P1−6). P1 and P1a had the same repeating unit but different molecular 29613


Research Article

ACS Applied Materials & Interfaces

Interestingly, all synthesized polymers (P1−6) could selfassemble into NPs without surfactants in water via nanoprecipitation method.50 Transmission electron microscopy (TEM) images in Figure 1 showed that spherical NPs were obtained. Diameters of NPs ranged from 100 to 250 nm, which were consistent with data in Table S1 measured by dynamic light scattering (DLS). Stability of NPs in water and Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) was investigated by DLS. The unchanged diameters and PDIs of NPs in Figure 2 validated that P1−6 NPs were stable in water for over 20 days. All NPs were stable in DMEM with 10% FBS for 24 h (Figure S3). The formed NPs are redox responsive because of the existence of disulfide bonds. The reduction-induced dissociation of NPs was monitored by DLS in the presence of 10 mM glutathione (GSH). To exclude the interference of molecular weight on redox sensitivity, P1a with similar molecular weight was synthesized for comparison. First, P1a could self-assemble into stable NPs, as revealed in Figure S4. Then, P1, P1a, P2, P4, P5, and P6 NPs with the same content of disulfide bonds were each treated by 10 mM GSH. The diameters of P1, P1a, and P2 NPs increased gently with time from 0.5 to 6 h (Figure 3A−C). However, the diameters and PDIs of P4−6 NPs increased quickly after treatment for 0.5 h (Figure 3D−F). As listed in Table S2, PDIs of P4−P6 NPs became closer to 1 and their diameters reached several microns. In addition, large aggregates could be seen for P4−P6 NPs in Figure S5, whereas P1 and P2 NPs appeared little muddy after 0.5 h. The dissociation of P1a NPs is similar to that of P1 NPs, as evidenced in Figure 3B and Table S2. Formation of precipitates was attributed to disassembly of NPs and rearrangement of hydrophobic debris. Molecular weight was not the reason for rapid dissociation to rearrange debris into large aggregates because P1, P1a, and P2 NPs had similar dissociation behavior.

weights. We shortened the reaction time to synthesize P1a. All polymers were characterized by proton nuclear magnetic resonance (1H NMR) and size-exclusion chromatography (SEC). We could clearly resolve all of the protons of the repeating units in Figure S1. The ratios of the integral area were consistent with theoretical value, which indicated the successful synthesis of all of the polymers. The number-average molecular weight (Mn), weight-average molecular weight (Mw), and dispersity (Đ) of polymers obtained by SEC are found in Table 1. The molecular weight of polymers decreased when πTable 1. SEC Results of P1−6 Synthesized via Multicomponent Passerini Reaction entry a

P1 P1ab P2a P3a P4a P5a P6c

Mn

Mw

Đd

15.4 2.25 6.25 3.00 3.94 3.10 3.63

24.1 2.78 9.95 3.65 5.58 3.76 4.98

1.58 1.25 1.59 1.21 1.41 1.21 1.37

a The reaction time was 4 days. bP1a and P1 had the same repeating unit, but P1a had low molecular weight because the reaction time was 12 h. cThe reaction time was 7 days. dĐ = Mw/Mn.

conjugated surface of aldehyde-containing molecules increased (Figure S2). From P1 to P6, Mn decreased from 15.4 to 3.6 kg mol−1, whereas polydispersity index (PDI) varied steadily in a certain range of 1.20−1.60. It seemed that π-conjugated surface affects the synthesis of polymers via the Passerini reaction because Mn of synthetic polymers decreased with an increase of π-conjugated surface.

Figure 1. TEM images of (A) P1, (B) P2, (C) P3, (D) P4, (E) P5, and (F) P6 NPs. The scale bar is 500 nm. 29614


Research Article


Figure 2. Stability of (A) P1, (B) P2, (C) P3, (D) P4, (E) P5, and (F) P6 NPs in water measured by DLS for about 20 days.

Figure 3. Reduction-induced disassembly led to size changes of (A) P1, (B) P2, (C) P3, (D) P4, (E) P5, and (F) P6 NPs. All NPs at the same concentration of disulfide bond were treated with 10 mM GSH under shaking at 37 °C.

500, and 562 nm in Figure 4A, respectively. The maximum absorption of the corresponding NPs shifted with 5, 8, and 11 nm, respectively. The bathochromic shift of absorption confirmed the formation of NPs.51 The maximum emission wavelength of P4, P5, and P6 in DMSO centered at 468, 525, and 570 nm, respectively. P4 NPs in water had similar maximum emission with P4 in DMSO. However, P5 and P6 NPs showed poor fluorescence in Figure 4B due to aggregation-caused quenching (ACQ).52 We took photos to visually observe the luminescent properties under 365 nm light irradiation, which are shown in Figure 4C. P3 NPs in water emitted strong blue fluorescence, but P3 in DMSO had no

Although molecular structure plays a key role in the stability of aggregates, Π-conjugated surface affected the speed of dissociation and rearrangement. The π-conjugated planes of P4, P5, and P6 NPs were larger than those of P1, P1a, and P2 NPs, so π−π interactions in P4, P5, and P6 NPs were stronger than those in P1, P1a, and P2 NPs. Strong π−π interactions led to rapid disintegration and reassembly of P4−6 NPs. Optical Properties of NPs. The photophysical properties were investigated by UV−vis absorption and photoluminescence emission for tetraphenylethene (TPE)-containing P3, coumarin (COU)-containing P4, and BODIPY-containing P5, P6, and their corresponding NPs. The maximum absorption of P4, P5, and P6 in dimethylsulfoxide (DMSO) peaked at 385, 29615


Research Article


Figure 4. (A) UV−vis absorption and (B) fluorescence spectra of P4−6 in DMSO and corresponding NPs in water. (C) Photos of all polymers in DMSO and all NPs in water before and after 365 nm light irradiation.

Figure 5. CLSM images of HeLa cells incubated with (A) P5 and (B) P6 NPs for 0.5 and 2 h. (A) Cell nuclei stained by DAPI showed blue fluorescence and P5 showed green fluorescence in cells. Their images and overlays of both images are shown from left to right. (B) P6 showed redblue fluorescence in cells. The images of cell nuclei (blue) and P6 (red) and overlays of both images are shown from left to right. The scale bar is 50 μm.

fluorescence of P3 NPs was absorbed by P5 NPs, which in turn emitted green fluorescence, as shown in Figure S6D. To our surprise, the mixture formed by directly mixing P3 NPs and P5 NPs immediately emitted green fluorescence (λem = 525 nm), as shown in Figure S8, confirming the FRET between P3 NPs and P5 NPs. Cellular Uptake and Bioimaging. The cytocompatibility of P1−6 NPs toward human cervical carcinoma (HeLa) cells was evaluated via methyl thiazolyl tetrazolium (MTT) assays. Cell viability was more than 80% when the cells were incubated with NPs (≥10 μg mL−1) for 24 h, as shown in Figure S9, indicating the good biocompatibility of P1−6 NPs. We used confocal laser scanning microscopy (CLSM) to study the cellular uptake of P4−6 NPs. The intensity of blue fluorescence in cytoplasm increased with time from 0.5 to 2 h, as shown in Figure S10, which indicated that P4 NPs could be endocytosed. To evaluate the cellular uptake and disassembly of P5 and P6 NPs, we stained cell nuclei by diamidino-phenyl-indole

fluorescence due to the aggregation-induced emission (AIE) effect.53 Fluorescence resonance energy transfer (FRET) effect was revealed between P3 and P5.54−59 The mixture of P3 and P5 was dissolved in tetrahydrofuran (THF) and then the solution was added dropwise into water with stirring. After THF was evaporated completely, the P3 + P5 NPs were obtained. The TEM image in Figure S6A showed that P3 + P5 could selfassemble into NPs in water. The NPs with 130 nm diameter kept stable in water over 2 weeks as evidenced by DLS in Figure S6B. Two peaks centered, respectively, at 314 and 508 nm in the UV−vis absorption spectra (Figure S7), validating that P3 + P5 NPs were successfully fabricated. Under 365 nm light irradiation, P3, P5, and P3 + P5 NPs emitted blue, no, and green fluorescence, respectively, as shown in Figure S6C. The maximum fluorescence wavelength of P3 and P3 + P5 NPs centered at 465 and 525 nm, respectively, whereas P5 NPs had no fluorescence under 360 nm excitation. The emitting 29616


Research Article


Figure 6. (A) TEM image of core−shell NPs co-assembled by P1 and PEG-PLA. The scale bar is 500 nm. (B) Cell viability of HeLa cells incubated with Taxol (Tax), PTX@P1-2-8 NPs, and PTX@P1-8-2 NPs at different PTX concentrations.

Figure 7. (A) Quantification of ICG degradation by ROS generated from P4 in DMSO and P4 NPs in water. The concentration of ICG was 10 μg mL−1. The concentration of P4 and P4 NPs was 40 μg mL−1. (B) Cell viability of HeLa cells treated with P4 NPs under blue-light irradiation for 0.5 h and cells treated with P4 NPs without irradiation as control.

PEG-PLA as the shell) because P1 was hydrophobic and PEGPLA was amphiphilic. TEM images in Figure 6A validated the formation of core−shell NPs. Inspired by the TEM images of core−shell NPs, we adjusted the weight rate of P1 and PEGPLA to encapsulate antitumor drug, paclitaxel (PTX). As shown in Figure S14 and Table S3, core−shell NPs with diameter between 100 and 200 nm were obtained by varying the weight rate of P1 and PEG-PLA. The drug loading increased from 0.63 to 4.11% and loading efficiency reached 13.7% from 2.1% as the weight rate of P1 and PEG-PLA increased from 0:10 to 8:2 (Table S3). The PTX-loading content and loading efficiency increased with the increase of hydrophobicity and π-conjugated planes of drug-delivery systems.50,60 Then, we evaluated cytotoxicity of PTX-loading NPs, as shown in Figures 6B and S15. All PTX-loading P1 and PEG-PLA NPs showed potent cytotoxicity toward HeLa cells. About 85% of cells were killed by 5 μg mL−1 PTX for NPs, which is comparable to the toxicity of Taxol (Tax). These results indicated that PTX-loading P1 and PEG-PLA NPs kept the antitumor activity. Phototoxicity. It was reported that coumarin could generate reactive oxygen species (ROS) to break ester bond under visible-light irradiation61 and be used for photodynamic therapy (PDT).62,63 We presumed that P4 NPs are potential

(DAPI). P5 and P6 NPs were successfully internalized and dissociated to emit green and red fluorescences, as shown in Figure 5A,B. The live cell imaging could be tuned with blue, green, or red fluorescence by varying the structure of polymers. Furthermore, the colocalization experiment indicated that P5 and P6 NPs were located in the lysosomes, as shown in Figures S11 and S12. The green and red fluorescences were coincident, which showed the good colocalization of P5 and P6 NPs and lysosomes.51 We chose P6 NPs to study the imaging ability in living animal. A Kunming mouse was intratumorally injected with P6 NPs (300 μg mL−1). After injection for 3 h, we could clearly observe bright fluorescence in tumor, as shown in Figure S13A. The fluorescence was visible after injection for 6 days (Figure S13D), which meant that P6 NPs could “light” tumor for long time. Comparing these images (Figure S13A−D), the fluorescence intensity decreased with time due to metabolism. Finally, heart, liver, spleen, lung, kidney, and tumor were separated for imaging. The tumor gave obvious fluorescence, and other organs had no fluorescence, as shown in Figure S13E. Drug Loading and Delivery. Polymer P1 could selfassemble into NPs in aqueous media. We hypothesized P1, and poly(ethylene glycol)2k-block-poly(D,L-lactide)2k (PEG-PLA) could co-assemble into core−shell NPs (P1 as the core, 29617


Research Article


Figure 8. TEM images of (A) P5-I and (B) P6-I NPs. The scale bar is 500 nm. (C) UV−vis absorption and (D) fluorescence spectra of P5-I, P6-I in DMSO and corresponding NPs in water.

red fluorescence was found in the experimental group (cells incubated with P4 NPs under irradiation), which confirmed that P4 NPs could kill cells under irradiation. Finally, we used 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as an ROS sensor to assess the intracellular ROS generation ability of P4 NPs. Oxidized DCFH (DCF) would emit green fluorescence. The experimental group showed stronger green fluorescence compared to the control groups, as shown in Figure S18. These results demonstrated that P4 NPs could produce ROS in HeLa cells and be used as photosensitizer for PDT. Iodinated BODIPYs could effectively produce ROS due to the heavy atom effect.65−67 As P5 and P6 contained BODIPY, we synthesized P5-I and P6-I NPs as shown in Scheme S1. The disappearance of the proton peaks (6.2 ppm for P5-I; 6.2 and 6.9 ppm for P6-I) in 1H NMR demonstrated that iodine atoms successfully replaced hydrogen atom to form P5-I and P6-I (Figures S19 and S1E,F). Both P5-I and P6-I could selfassemble into NPs with diameter of about 200 nm, as shown in Figure 8A,B. P5-I and P6-I NPs are stable in aqueous media for about 20 days and in DMEM with 10% FBS for 24 h according to unchanged size in Figure S20. As shown in Figure 8C, the maximum absorption of P5-I and P6-I in DMSO peaked at 535 and 580 nm, respectively. Furthermore, the maximum absorption of the corresponding NPs in water was red-shifted with 9 and 6 nm, respectively. The maximum emission

photosensitizers for PDT. First, we evaluated the ROS generation ability of P4 and P4 NPs using indocyanine green (ICG) as scavenger.64 After irradiation for 5 min under blue light (wavelength = 450 nm, 10 mW cm−2), the absorbance intensity of ICG (λex = 790 nm) was unchanged, as shown in Figure S16A,C. However, the absorbance reduced continually in the presence of P4 in DMF or P4 NPs in water under irradiation (Figure S16B,D). P4 in DMF and P4 NPs in water possess ability to produce ROS and degrade ICG. As shown in Figure 7A, almost 40% of ICG was degraded by ROS generated from P4 and P4 NPs under blue-light irradiation for 5 min. Then, we evaluated the PDT effect of P4 NPs on HeLa cells. HeLa cells under irradiation without P4 NPs grew normally, and the cell viability did not change compared to the cells without irradiation, as shown in Figure 7B. The cell viability of HeLa cells incubated with P4 NPs but without irradiation did not decrease, either. However, more than 60% of cells incubated with P4 NPs (45 μg mL−1) were killed upon irradiation for 30 min (Figure 7B). To visually observe the PDT effect of P4 NPs, we used live/ dead staining to distinguish the live and dead cells. Live cells were stained with calcein-AM to emit green fluorescence, and dead/late apoptotic (red) cells were stained with propidium iodide (PI) to emit red fluorescence. As shown in Figure S17, only green fluorescence was observed in all control groups and 29618


Research Article


Figure 9. (A) Quantification of ICG degradation by ROS generated from P5-I and P6-I NPs in water. The concentration of ICG was 10 μg mL−1. The concentration of P4 and P5-I and P6-I NPs was 10 μg mL−1. Cell viability of HeLa cells treated with (B) P5-I and (C) P6-I NPs under greenlight irradiation for 0.5 h; cells treated with P5-I or P6-I NPs without irradiation are controls.

Figure 10. (A) Tumor volumes of mice in 10 days. (B) Tumor weight on the 10th day. (C) H&E staining of heart, liver, spleen, lung, and kidney on the 10th day. The scale bar is 100 μm. * P < 0.05, n.s. P > 0.05.

quickly in the presence of P5-I, P6-I, and corresponding NPs under irradiation (Figure S21B,C,E,F), demonstrating that P5I, P6-I, and corresponding NPs possess ability to generate ROS upon irradiation. As shown in Figures 9A and S22, more than 70% of ICG was degraded under green-light irradiation for 2.5 min with P5-I and P6-I in DMF or corresponding NPs in water. The intracellular uptake of P5-I and P6-I NPs toward HeLa cells was observed by CLSM (Figure S23). Green fluorescence from P5-I in Figure S23A and red fluorescence from P6-I in

wavelength of P5-I and P6-I in DMSO centered at 564 and 600 nm in Figure 8D. The bathochromic shifts of absorption and emission bands comparing to those of P5 and P6 further proved the successful synthesis of P5-I and P6-I. As shown in Figure S21, ROS generation of P5-I, P6-I, and corresponding NPs was evaluated via degradation of ICG. Little variation in the absorbance intensity of ICG under green-light irradiation (560 nm, 10 mW cm−2) for 2.5 min showed that irradiation did not degrade ICG in DMF and water (Figure S21A,D). However, the absorbance intensity of ICG reduced 29619


Research Article


Eagle’s medium containing 10% fetal bovine serum was purchased from GIBCO. Dimethylsulfoxide-d6 (DMSO-d6) and chloroform-d (CDCl3) were purchased from Cambridge Isotope Laboratories, Inc. Characterizations. AV-400 NMR spectrometer (Bruker) was used for recording 1H NMR spectra in CDCl3 and DMSO-d6. A DLS instrument (Malvern Zetasizer Nano) was used to measure diameter and size distribution. A transmission electron microscope (JEOL JEM1011) was used to measure the morphology of the NPs. To prepare TEM samples, a drop of NPs in water (1 μg/mL) was deposited onto a copper grid with a carbon coating. The specimens were air-dried and measured at room temperature. The PDIs of the polymer were measured by SEC on a TOSOH HLC-8220 SEC instrument (column: Super HZM-H×3) at 40 °C using tetrahydrofuran (THF) as eluent with a flowing rate of 0.35 mL/min. Statistical significance analysis was assessed by SPSS via one-way ANOVA test. N.s. meant no difference, but *P < 0.05 was considered statistically highly significant. Synthesis and Preparation of P1−6 NPs, Cellular Imaging, and Cytotoxicity Experiments. The detailed data are presented in Supporting Information. ROS Detection in Cells. The experiment was done according to the specification of ROS detection kit (S0033), which was purchased from Beyotime. Live/Dead Staining. The experiment was done according to the specification of cell viability assay kit (KGAF001), which was purchased from KeyGEN BioTECH. Fluorescence Images in Vivo. All animal experiments were carried out in accordance with the regulation for Research Animals from Jilin University Studies Committee. A female Kunming mouse was intratumorally injected with P6 NPs (200 μg mL−1, 300 μL). In vivo fluorescence imaging was carried out by fluorescence imaging system (CRI Maestro 500FL) after the mouse was anesthetized. Tumor Inhabitation in Vivo. Female Kunming mice with U14 tumor in the armpit of the left anterior limb were used. The mice were divided into four groups: (1) control group (Con) treated with saline; (2) saline + light; (3) P6-I NPs; and (4) P6-I NPs + light. The mice were intratumorally injected with P6-I NPs (300 μg mL−1, 200 μL). After 12 h, the mice were irradiated with green light (150 mW; diameter of light spot, 5 mm) for 0.5 h. These processes were carried out every 36 h for three times. Tumor volume and body weight were measured every 2 days.

Figure S23B demonstrated that both P5-I and P6-I NPs could be internalized and emitted fluorescence. Using DCFH-DA as an ROS sensor, the intracellular ROS generation ability of P5-I and P6-I NPs was confirmed by stronger green fluorescence compared to the control groups under green-light irradiation in Figure S24. Furthermore, the phototoxicities of P5-I and P6-I NPs toward HeLa cells were studied by MTT assays, as shown in Figure 9B,C. There was no toxicity when cells were only incubated with both NPs at a maximum concentration of 10 μg mL−1. Green-light irradiation (10 mW cm−2) could not kill cells, either. However, under irradiation for 30 min, more than 80% of cells were dead when the concentration of NPs was 2.5 μg mL−1. Live/dead staining was also used to confirm the PDT effect of both NPs. As shown in Figure S25, we could not observe red fluorescence in all control groups, but red fluorescence could be found everywhere in the experimental groups (cells incubated with both NPs under irradiation). These results spurred us to study PDT of P5-I or P6-I NPs in living animal. We chose P6-I NPs for the tumor treatment in mice. Kunming mice with mouse uterine cervical cancer cells were intratumorally injected with P6-I NPs (300 μg mL−1, 200 μL). The mice were treated with green-light irradiation for 0.5 h. We weighed the mice and measured the tumor sizes every other day. Tumor growth in the experiment group was successfully inhibited (P6-I NPs + light) and showed significant difference with those in control groups (Figure 10A). After 10 days, tumors and main organs were separated for further characterization. Tumors in the experiment group were smaller than those in control groups, as shown in Figure S26A. Average tumor weight of the control group treated with saline was 2 g, which was 4 times heavier than that of the experiment group (Figure 10B). The body weight of all mice showed a little increase (Figure S26B), which demonstrated that treatment of P6-I NPs and irradiation were not toxic to mice. Finally, main organs were stained with hematoxylin and eosin (H&E). Heart, liver, spleen, lung, and kidney of all mice were normal compared with the control group treated with saline (Figure 10C). These results demonstrated that P6-I NPs could be used as potential photosensitizers for PDT.

■

S Supporting Information *

■

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b10763. Experimental method for the synthesis of all polymers; preparation of all NPs and cell experiments; characterization of all NPs by DLS; 1H NMR spectroscopy; characterization of FRET; cell viability of HeLa cells incubated with all NPs; CLSM images of HeLa cells incubated with NPs; colocalization of P5, P6, and lysosomes; fluorescence images in vivo; characterization of producing ROS; photos of tumors; and body weight of mice (Scheme S1, Tables S1−S3, Figures S1−S26) (PDF)

CONCLUSIONS In summary, we have prepared versatile polymers containing disulfide bonds, imaging agents, and photosensitizers via multicomponent Passerini reaction. The formed polymers were not only redox sensitive, but also showed great potential for bioapplication. All polymer NPs were stable in water and showed various optical properties like AIE, ACQ, and FRET. Their biological applications were explored in detail due to their good cytocompatibility. The NPs possessed different biological functions, such as drug delivery, multicolored cellular imaging, and photodynamic therapy (PDT). We believe multicomponent Passerini reaction as an atom-economic and one-step way that shows great potential to synthesize multifunctional polymers for preparing nanoparticles, as well as versatile biological application, especially theranostics.

■

ASSOCIATED CONTENT

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

EXPERIMENTAL METHODS

ORCID

Materials. 3,3-Dithiodipropionic acid and o-nitrobenzaldehyde were purchased from Shanghai Sun Chemical Technology Co., Ltd. 1,6-Diisocyanohexane was purchased from Aldrich. GSH was purchased from Shanghai Yuanye Biological Technology Co., Ltd. 1Naphthaldehyde was purchased from Aladdin. Dulbecco’s modified

Zhigang Xie: 0000-0003-2974-1825 Notes

The authors declare no competing financial interest. 29620


Research Article


■

(20) Li, L.; Lv, A.; Deng, X.-X.; Du, F.-S.; Li, Z.-C. Facile Synthesis of Photo-Cleavable Polymers via Passerini Reaction. Chem. Commun. 2013, 49, 8549−8551. (21) Lin, W.; Sun, T.; Zheng, M.; Xie, Z.; Huang, Y.; Jing, X. Synthesis of Cross-Linked Polymers via Multi-Component Passerini Reaction and Their Application as Efficient Photocatalysts. RSC Adv. 2014, 4, 25114−25117. (22) Lin, W.; Guan, X.; Sun, T.; Huang, Y.; Jing, X.; Xie, Z. Reduction-Sensitive Amphiphilic Copolymers Made via MultiComponent Passerini Reaction for Drug Delivery. Colloids Surf., B 2015, 126, 217−223. (23) Lin, W.; Li, Y.; Zhang, W.; Liu, S.; Xie, Z.; Jing, X. Near-Infrared Polymeric Nanoparticles with High Content of Cyanine for Bimodal Imaging and Photothermal Therapy. ACS Appl. Mater. Interfaces 2016, 8, 24426−24432. (24) Chen, G.; Roy, I.; Yang, C.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826−2885. (25) Alonso-Cristobal, P.; Vilela, P.; El-Sagheer, A.; Lopez-Cabarcos, E.; Brown, T.; Muskens, O. L.; Rubio-Retama, J.; Kanaras, A. G. Highly Sensitive DNA Sensor Based on Upconversion Nanoparticles and Graphene Oxide. ACS Appl. Mater. Interfaces 2015, 7, 12422− 12429. (26) Caminade, A. M. Inorganic Dendrimers: Recent Advances for Catalysis, Nanomaterials, and Nanomedicine. Chem. Soc. Rev. 2016, 45, 5174−5186. (27) Krol, S.; Macrez, R.; Docagne, F.; Defer, G.; Laurent, S.; Rahman, M.; Hajipour, M. J.; Kehoe, P. G.; Mahmoudi, M. Therapeutic Benefits from Nanoparticles: The Potential Significance of Nanoscience in Diseases with Compromise to The Blood Brain Barrier. Chem. Rev. 2013, 113, 1877−1903. (28) Howes, P. D.; Chandrawati, R.; Stevens, M. M. Bionanotechnology. Colloidal Nanoparticles as Advanced Biological Sensors. Science 2014, 346, No. 1247390. (29) Skeete, Z.; Cheng, H.; Crew, E.; Lin, L.; Zhao, W.; Joseph, P.; Shan, S.; Cronk, H.; Luo, J.; Li, Y.; Zhang, Q.; Zhong, C.-J. Design of Functional Nanoparticles and Assemblies for Theranostic Applications. ACS Appl. Mater. Interfaces 2014, 6, 21752−21768. (30) Søndergaard, R. V.; Christensen, N. M.; Henriksen, J. R.; Kumar, E. K.; Almdal, K.; Andresen, T. L. Facing the Design Challenges of Particle-Based Nanosensors for Metabolite Quantification in Living Cells. Chem. Rev. 2015, 115, 8344−8378. (31) Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence. Chem. Rev. 2015, 115, 10530−10574. (32) Ma, Y.-C.; Wang, J.-X.; Tao, W.; Sun, C.-Y.; Wang, Y.-C.; Li, D.D.; Fan, F.; Qian, H.-S.; Yang, X.-Z. Redox-Responsive Polyphosphoester-Based Micellar Nanomedicines for Overriding Chemoresistance in Breast Cancer Cells. ACS Appl. Mater. Interfaces 2015, 7, 26315−26325. (33) Lim, E.-K.; Kim, T.; Paik, S.; Haam, S.; Huh, Y.-M.; Lee, K. Nanomaterials for Theranostics: Recent Advances and Future Challenges. Chem. Rev. 2015, 115, 327−394. (34) He, C.; Liu, D.; Lin, W. Nanomedicine Applications of Hybrid Nanomaterials Built from Metal−Ligand Coordination Bonds: Nanoscale Metal−Organic Frameworks and Nanoscale Coordination Polymers. Chem. Rev. 2015, 115, 11079−11108. (35) Min, Y.; Caster, J. M.; Eblan, M. J.; Wang, A. Z. Clinical Translation of Nanomedicine. Chem. Rev. 2015, 115, 11147−11190. (36) Wang, H.; Wu, J.; Xie, K.; Fang, T.; Chen, C.; Xie, H.; Zhou, L.; Zheng, S. Precise Engineering of Prodrug Cocktails into Single Polymeric Nanoparticles for Combination Cancer Therapy: Extended and Sequentially Controllable Drug Release. ACS Appl. Mater. Interfaces 2017, 9, 10567−10576. (37) Wang, H.; Xie, H.; Wu, J.; Wei, X.; Zhou, L.; Xu, X.; Zheng, S. Structure-Based Rational Design of Prodrugs To Enable Their Combination with Polymeric Nanoparticle Delivery Platforms for

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Project Nos. 51522307 and 81673396). The authors thank Qi Zhang for quantification of PTX by LCMS/MS.

■

REFERENCES

(1) Brauch, S.; van Berkel, S. S.; Westermann, B. Higher-Order Multicomponent Reactions: beyond Four Reactants. Chem. Soc. Rev. 2013, 42, 4948−4962. (2) Liu, Y.; Gao, M.; Lam, J. W. Y.; Hu, R.; Tang, B. Z. CopperCatalyzed Polycoupling of Diynes, Primary Amines, and Aldehydes: A New One-Pot Multicomponent Polymerization Tool to Functional Polymers. Macromolecules 2014, 47, 4908−4919. (3) Yang, B.; Zhao, Y.; Wang, S.; Zhang, Y.; Fu, C.; Wei, Y.; Tao, L. Synthesis of Multifunctional Polymers through the Ugi Reaction for Protein Conjugation. Macromolecules 2014, 47, 5607−5612. (4) Xue, H.; Zhao, Y.; Wu, H.; Wang, Z.; Yang, B.; Wei, Y.; Wang, Z.; Tao, L. Multicomponent Combinatorial Polymerization via the Biginelli Reaction. J. Am. Chem. Soc. 2016, 138, 8690−8693. (5) Ramírez-Ornelas, D. E.; Alvarado-Martinez, E.; Banuelos, J.; Lopez Arbeloa, I.; Arbeloa, T.; Mora-Montes, H. M.; Perez-Garcia, L. A.; Pena-Cabrera, E. FormylBODIPYs: Privileged Building Blocks for Multicomponent Reactions. The Case of the Passerini Reaction. J. Org. Chem. 2016, 81, 2888−2898. (6) Rezaei, A.; Akhavan, O.; Hashemi, E.; Shamsara, M. Toward Chemical Perfection of Graphene-Based Gene Carrier via Ugi Multicomponent Assembly Process. Biomacromolecules 2016, 17, 2963−2971. (7) Kayser, L. V.; Vollmer, M.; Welnhofer, M.; Krikcziokat, H.; Meerholz, K.; Arndtsen, B. A. Metal-Free, Multicomponent Synthesis of Pyrrole-Based pi-Conjugated Polymers from Imines, Acid Chlorides, and Alkynes. J. Am. Chem. Soc. 2016, 138, 10516−10521. (8) Dömling, A.; Wang, W.; Wang, K. Chemistry and Biology Of Multicomponent Reactions. Chem. Rev. 2012, 112, 3083−3135. (9) Passerini, M. Isonitriles. II. Compounds with Aldehydes or with Ketones and Monobasic Organic Acids. Gazz. Chim. Ital. 1921, 51, 126−129. (10) Dö mling, A. Recent Developments in Isocyanide Based Multicomponent Reactions in Applied Chemistry. Chem. Rev. 2006, 106, 17−89. (11) Sehlinger, A.; Kreye, O.; Meier, M. A. R. Tunable Polymers Obtained from Passerini Multicomponent Reaction Derived Acrylate Monomers. Macromolecules 2013, 46, 6031−6037. (12) Deng, X.-X.; Li, L.; Li, Z.-L.; Lv, A.; Du, F.-S.; Li, Z.-C. Sequence Regulated Poly(ester-amide)s Based on Passerini Reaction. ACS Macro Lett. 2012, 1, 1300−1303. (13) Dö mling, A.; Ugi, I. Multicomponent Reactions with Isocyanides. Angew. Chem., Int. Ed. 2000, 39, 3168−3210. (14) Kreye, O.; Toth, T.; Meier, M. A. Introducing Multicomponent Reactions to Polymer Science: Passerini Reactions of Renewable Monomers. J. Am. Chem. Soc. 2011, 133, 1790−1792. (15) Kakuchi, R. Multicomponent Reactions in Polymer Synthesis. Angew. Chem., Int. Ed. 2014, 53, 46−48. (16) Solleder, S. C.; Meier, M. A. R. Sequence Control in Polymer Chemistry through the Passerini Three-Component Reaction. Angew. Chem., Int. Ed. 2014, 53, 711−714. (17) Solleder, S. C.; Zengel, D.; Wetzel, K. S.; Meier, M. A. R. A Scalable and High-Yield Strategy for the Synthesis of SequenceDefined Macromolecules. Angew. Chem., Int. Ed. 2016, 55, 1204−1207. (18) Zhang, J.; Zhang, M.; Du, F.-S.; Li, Z.-C. Synthesis of Functional Polycaprolactones via Passerini Multicomponent Polymerization of 6Oxohexanoic Acid and Isocyanides. Macromolecules 2016, 49, 2592− 2600. (19) Lv, A.; Deng, X.-X.; Li, L.; Li, Z.-L.; Wang, Y.-Z.; Du, F.-S.; Li, Z.-C. Facile Synthesis of Multi-Block Copolymers Containing Poly(ester−amide) Segments with An Ordered Side Group Sequence. Polym. Chem. 2013, 4, 3659−3662. 29621


Research Article

ACS Applied Materials & Interfaces Enhanced Antitumor Efficacy. Angew. Chem., Int. Ed. 2014, 126, 11716−11721. (38) Wang, H.; Xie, H.; Wang, J.; Wu, J.; Ma, X.; Li, L.; Wei, X.; Ling, Q.; Song, P.; Zhou, L.; Xu, X.; Zheng, S. Self-Assembling Prodrugs by Precise Programming of Molecular Structures that Contribute Distinct Stability, Pharmacokinetics, and Antitumor Efficacy. Adv. Funct. Mater. 2015, 25, 4956−4965. (39) Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, Functionalization Strategies and Biomedical Applications of Targeted Biodegradable/Biocompatible Polymer-Based Nanocarriers For Drug Delivery. Chem. Soc. Rev. 2013, 42, 1147−1235. (40) Cui, J.; Richardson, J. J.; Björnmalm, M.; Faria, M.; Caruso, F. Nanoengineered Templated Polymer Particles: Navigating the Biological Realm. Acc. Chem. Res. 2016, 49, 1139−1148. (41) Kowalczuk, A.; Trzcinska, R.; Trzebicka, B.; Müller, A. H. E.; Dworak, A.; Tsvetanov, C. B. Loading of Polymer Nanocarriers: Factors, Mechanisms and Applications. Prog. Polym. Sci. 2014, 39, 43− 86. (42) Wu, J.; Zhang, J.; Deng, C.; Meng, F.; Cheng, R.; Zhong, Z. Robust, Responsive, and Targeted PLGA Anticancer Nanomedicines by Combination of Reductively Cleavable Surfactant and Covalent Hyaluronic Acid Coating. ACS Appl. Mater. Interfaces 2017, 9, 3985− 3994. (43) Blum, A. P.; Kammeyer, J. K.; Rush, A. M.; Callmann, C. E.; Hahn, M. E.; Gianneschi, N. C. Stimuli-Responsive Nanomaterials for Biomedical Applications. J. Am. Chem. Soc. 2015, 137, 2140−2154. (44) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991−1003. (45) Lu, Y.; Aimetti, A. A.; Langer, R.; Gu, Z. Bioresponsive Materials. Nat. Rev. Mater. 2016, 2, 16075−16091. (46) Lin, W.; Sun, T.; Xie, Z.; Gu, J.; Jing, X. A Dual-Responsive Nanocapsule via Disulfide-Induced Self-Assembly for Therapeutic Agent Delivery. Chem. Sci. 2016, 7, 1846−1852. (47) Zhang, W.; Lin, W.; Pei, Q.; Hu, X.; Xie, Z.; Jing, X. RedoxHypersensitive Organic Nanoparticles for Selective Treatment of Cancer Cells. Chem. Mater. 2016, 28, 4440−4446. (48) Wang, Y.-Z.; Deng, X.-X.; Li, L.; Li, Z.-L.; Du, F.-S.; Li, Z.-C. One-Pot Synthesis of Polyamides with Various Functional Side Groups via Passerini Reaction. Polym. Chem. 2013, 4, 444−448. (49) Deng, X.-X.; Du, F.-S.; Li, Z.-C. Combination of Orthogonal ABB and ABC Multicomponent Reactions toward Efficient Divergent Synthesis of Dendrimers with Structural Diversity. ACS Macro Lett. 2014, 3, 667−670. (50) Wu, J.; Zhao, L.; Xu, X.; Bertrand, N.; Choi, W., II; Yameen, B.; Shi, J.; Shah, V.; Mulvale, M.; MacLean, J. L.; Farokhzad, O. C. Hydrophobic Cysteine Poly(disulfide)-based Redox-Hypersensitive Nanoparticle Platform for Cancer Theranostics. Angew. Chem., Int. Ed. 2015, 54, 9218−9223. (51) Lin, W.; Zhang, W.; Sun, T.; Gu, J.; Xie, Z.; Jing, X. Effect of Molecular Structure on Stability of Organic Nanoparticles Formed by Bodipy Dimers. Langmuir 2016, 32, 9575−9581. (52) Ren, C.; Wang, H.; Mao, D.; Zhang, X.; Fengzhao, Q.; Shi, Y.; Ding, D.; Kong, D.; Wang, L.; Yang, Z. When Molecular Probes Meet Self-Assembly: An Enhanced Quenching Effect. Angew. Chem., Int. Ed. 2015, 54, 4823−4827. (53) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718−11940. (54) Cheng, D.; Pan, Y.; Wang, L.; Zeng, Z.; Yuan, L.; Zhang, X.; Chang, Y.-T. Selective Visualization of the Endogenous Peroxynitrite in an Inflamed Mouse Model by a Mitochondria-Targetable TwoPhoton Ratiometric Fluorescent Probe. J. Am. Chem. Soc. 2017, 139, 285−292. (55) Liu, Y.; Qu, X.; Guo, Q.; Sun, Q.; Huang, X. QD-BiopolymerTSPP Assembly as Efficient BiFRET Sensor for Ratiometric and Visual Detection of Zinc Ion. ACS Appl. Mater. Interfaces 2017, 9, 4725− 4732. (56) Wu, Y.; Liu, J.; Wang, Y.; Li, K.; Li, L.; Xu, J.; Wu, D. Novel Ratiometric Fluorescent Nanothermometers Based on Fluorophores-

Labeled Short Single-Stranded DNA. ACS Appl. Mater. Interfaces 2017, 9, 11073−11081. (57) Lin, M.; Gao, Y.; Diefenbach, T. J.; Shen, J. K.; Hornicek, F. J.; Park, Y. I.; Xu, F.; Lu, T. J.; Amiji, M.; Duan, Z. Facial Layer-by-Layer Engineering of Upconversion Nanoparticles for Gene Delivery: NearInfrared-Initiated Fluorescence Resonance Energy Transfer Tracking and Overcoming Drug Resistance in Ovarian Cancer. ACS Appl. Mater. Interfaces 2017, 9, 7941−7949. (58) Yang, H. Y.; Fu, Y.; Jang, M.-S.; Li, Y.; Lee, J. H.; Chae, H.; Lee, D. S. Multifunctional Polymer Ligand Interface CdZnSeS/ZnS Quantum Dot/Cy3-Labeled Protein Pairs as Sensitive FRET Sensors. ACS Appl. Mater. Interfaces 2016, 8, 35021−35032. (59) Yang, L.; Cui, C.; Wang, L.; Lei, J.; Zhang, J. Dual-Shell Fluorescent Nanoparticles for Self-Monitoring of pH-Responsive Molecule-Releasing in a Visualized Way. ACS Appl. Mater. Interfaces 2016, 8, 19084−19091. (60) Hu, X.; Li, J.; Lin, W.; Huang, Y.; Jing, X.; Xie, Z. Paclitaxel Prodrug Nanoparticles Combining Chemical Conjugation and Physical Entrapment for Enhanced Antitumor Efficacy. RSC Adv. 2014, 4, 38405−39411. (61) Szymański, W.; Velema, W. A.; Feringa, B. L. Photocaging of Carboxylic Acids: A Modular Approach. Angew. Chem., Int. Ed. 2014, 53, 8682−8686. (62) Zou, Q.; Fang, Y.; Zhao, Y.; Zhao, H.; Wang, Y.; Gu, Y.; Wu, F. Synthesis and In Vitro Photocytotoxicity of Coumarin Derivatives for One- and Two-Photon Excited Photodynamic Therapy. J. Med. Chem. 2013, 56, 5288−5294. (63) Gangopadhyay, M.; Singh, T.; Behara, K. K.; Karwa, S.; Ghosh, S. K.; Singh, N. D. Coumarin-Containing-Star-Shaped 4-Arm-Polyethylene Glycol: Targeted Fluorescent Organic Nanoparticles for Dual Treatment of Photodynamic Therapy and Chemotherapy. Photochem. Photobiol. Sci. 2015, 14, 1329−1336. (64) Tang, C.-Y.; Wu, F.-Y.; Yang, M.-K.; Guo, Y.-M.; Lu, G.-H.; Yang, Y.-H. A Classic Near-Infrared Probe Indocyanine Green for Detecting Singlet Oxygen. Int. J. Mol. Sci. 2016, 17, 219−226. (65) Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.; Burgess, K. BODIPY Dyes in Photodynamic Therapy. Chem. Soc. Rev. 2013, 42, 77−88. (66) Huang, L.; Li, Z.; Zhao, Y.; Zhang, Y.; Wu, S.; Zhao, J.; Han, G. Ultralow-Power Near Infrared Lamp Light Operable Targeted Organic Nanoparticle Photodynamic Therapy. J. Am. Chem. Soc. 2016, 138, 14586−14591. (67) Yogo, T.; Urano, Y.; Ishitsuka, Y.; Maniwa, F.; Nagano, T. Highly Efficient and Photostable Photosensitizer Based on BODIPY Chromophore. J. Am. Chem. Soc. 2005, 127, 12162−12163.

29622


Rational Design of Polymeric Nanoparticles with Tailorable

Recommend Documents