AIE Supramolecular Assembly with FRET Effect for Visualizing Drug

Jun 18, 2019 - Here, we constructed a nanostructured pH/redox dual-responsive supramolecular drug carrier with both aggregation-induced emission (AIE)...
0 downloads 0 Views 4MB Size
Download PDF
Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

www.acsami.org

AIE Supramolecular Assembly with FRET Effect for Visualizing Drug Delivery Zhenzhen Dong,† Yanze Bi,‡ Hanrui Cui,† Yandong Wang,† Chunlei Wang,† Yan Li,*,‡ Hongwei Jin,*,§ and Caiqi Wang*,† †

School of Chemical Sciences, University of Chinese Academy of Sciences, Beijing 100049, China School of Materials Science and Engineering, Beihang University, Beijing 100083, China § State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Downloaded via BUFFALO STATE on July 17, 2019 at 14:00:33 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Here, we constructed a nanostructured pH/redox dual-responsive supramolecular drug carrier with both aggregationinduced emission (AIE) and Forster resonance energy transfer (FRET) effects, which enabled selective drug release and monitoring drug delivery and release processes. Taking the hyperbranched polyamide amine (H-PAMAM) with intrinsic AIE effects as the core, poly(ethylene glycol) (PEG) was bridged on its periphery by dithiodipropionic acid. Then, through the host−guest interaction of PEG and α-cyclodextrin, the supramolecular nanoparticles with AIE effects were constructed to load the anticancer drug doxorubicin (DOX). The supramolecular assembly has sufficiently large DOX loading due to the abundant cavities formed by branched structures. The hyperbranched core H-PAMAM has strong fluorescence, and the dynamic track of drug carriers and the dynamic drug release process can be monitored by the AIE and FRET effects between HPAMAM and DOX, respectively. Furthermore, the introduction of disulfide bonds and the pH sensitivity of H-PAMAM enable the achievement of rapid selective release of loaded DOX at the tumor while remaining stable under normal physiological conditions. In vitro cytotoxicity indicates that the drug-loaded supramolecular assembly has a good therapeutic effect on cancer. In addition, the H-PAMAM core is different from the traditional AIE functional group, which has no conjugated structure, such as a benzene ring, thereby providing better biocompatibility. This technology will have broad applications as a new drug delivery system. KEYWORDS: aggregation-induced emission, Forster resonance energy transfer, supramolecular assembly, drug delivery, drug release

1. INTRODUCTION Since the enhanced permeation and retention (EPR) effect improves the pharmacokinetics and pharmacodynamics, nanoparticle drug carriers can be selectively distributed in tumor tissues to increase the efficacy and reduce systemic toxicity and have been widely used in cancer treatment.1−3 Among them, the supramolecular nanodrug delivery systems constructed by noncovalent bonds have attracted much attention on account of their simple implementation methods and improvement of drug solubility and stability.4−7 At the same time, the use of responsive nanodrug delivery systems to achieve selective drug release has become a research hotspot.8−12 However, the nanoparticles are usually invisible, and the dynamic track cannot be observed. The aggregation-induced emission (AIE) effect proposed by Tang13 possesses obvious strengths such as no self-quenching and good light stability and light response to analytes. Its application in biological imaging and sensing has quickly attracted the attention of many researchers.14−18 The AIE effect © 2019 American Chemical Society

provides a viable idea for the visualization of nanodrug delivery systems. For example, Zhuang et al.19 fabricated a tetraphenylethene (TPE)-based polymeric micelle system for responsive drug release and cell imaging. Yu et al.20 constructed a pillar[5]arene-based [2]rotaxane based on TPE, which could light up mitochondria specifically and deliver anticancer drug. In addition to the typical AIE molecules such as TPE, other types of macromolecules containing only the auxochrome such as carbonyl groups, ester groups, amides, and aliphatic amines can also have fluorescent emission under appropriate conditions and exhibit an AIE effect.21−25 Although the fluorescence quantum yield of such materials is lower than that of conjugated polymer luminescent materials, these compounds are more similar to biological materials such as proteins and polysaccharides, exhibiting superior biocompatibility, lower cytotoxReceived: March 19, 2019 Accepted: June 18, 2019 Published: June 18, 2019 23840

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

Research Article

ACS Applied Materials & Interfaces

Figure 1. (a) Synthesis route of supramolecular assembly used to load DOX. (b) Schematic illustration of a pH/redox dual-responsive supramolecular assembly with the AIE and FRET effects for drug delivery and release.

icity, and greater endocytosis.26,27 Among them, hyperbranched polyamide amine (H-PAMAM) is a class of highly regarded unconventional luminescent macromolecules whose fluorescence source is attributed to the oxidation of tertiary amines.28−30 H-PAMAM has good biocompatibility, a large number of cavities formed by branched structures, and terminal amino groups, so it has great application potential in chemical modification31 and drug delivery.32 Appropriate modification of H-PAMAM will enhance its fluorescence, which opens the door for biological imaging of polymers based on H-PAMAM.33,34 In previous work,34 we grafted peptide chains at the outer end of HPAMAM to synthesize a hyperbranched polymeric peptide. As a result, the polymer exhibited good antibacterial and bacterial imaging capabilities for the monitoring antibacterial process in real time. Although the dynamic track of drug carriers in the cell can be tracked by the AIE effect, their release behavior remains unknown. Forster resonance energy transfer (FRET) is a method used to monitor the dynamic processes of specific imaging systems. The FRET effect between the nanocarrier and the drug can be utilized to study the release process of the carrier within the cell. When the drug is released, the FRET effect between carriers and loaded drug disappears, so that the mechanism of action is more clearly understood to achieve a better cancer treatment effect.35,36 Therefore, utilizing the host− guest interaction between poly(ethylene glycol) (PEG) and αcyclodextrin (α-CD), this study intended to fabricate a nanostructured pH/redox dual-responsive supramolecular drug carrier H-PAMAM-ss-mPEG/α-CD (HG/CD) with both AIE and FRET effects, used to visualize drug delivery and release processes, as shown in Figure 1. Taking the easily synthesized H-PAMAM with an intrinsic AIE effect as the core, PEG was bridged on its periphery by dithiodipropionic acid. Then, through the host−guest interaction of PEG and αcyclodextrin, the supramolecular nanoparticles exhibiting the

AIE effect were constructed for loading the anticancer drug doxorubicin (DOX) (HG/CD@DOX). The hyperbranched core H-PAMAM has intrinsic AIE effects, so the complex is free from the labored treatment of fluorescent dye labeling, and the monitoring of dynamic track of the drug can be realized. Importantly, the dynamic release process of the drug can be monitored through the FRET effect of HG/CD (donor) and DOX (receptor). In addition, the pH/redox dual responsiveness of HG/CD with excellent biocompatibility allows for a rapid selective release of loaded DOX in cancer cells. This work provides an easy, economical, and rapid way to build a nanodrug-loading platform that enables drug carrier tracing, dynamic drug release monitoring, responsive drug release, and chemotherapy.

2. EXPERIMENTAL SECTION 2.1. Materials and Characterizations. See the Supporting Information. 2.2. Synthesis of H-PAMAM. Referring to our previous work,34 the synthesis of H-PAMAM is as follows. Briefly, under a nitrogen atmosphere, 5.15 g of diethylenetriamine and 5.15 g of methyl acrylate were dissolved in methanol. The mixed solution was first reacted for 2 h in an ice water bath and then reacted at 25 °C for 2 d. Subsequently, the mixed solution was reacted at 60, 80, 100, and 120 °C for 1, 1, 1, and 2 h in vacuum, respectively. Final precipitation by diethyl ether gave a yellow viscous product. 2.3. Synthesis of mPEG-SS-COOH (Scheme S1, Supporting Information). The mPEG-SS-COOH is synthesized through esterification of poly(ethylene glycol) methyl ether (mPEG) and 3,3′-dithiodipropionic acid (DTDPA). Eight grams of mPEG, 1.58 g of DTDPA, 0.49 g of 4-(dimethylamino)pyridine, 1.65 g of N,N′dicyclohexylcarbodiimide, and 0.41 g of triethylamine were dissolved in anhydrous tetrahydrofuran and stirred for 2 d at 25 °C. The resulting white precipitate was filtered out. The remaining clear solution was precipitated through ice diethyl ether to give the product. 2.4. Synthesis of HG. In 10 mL of dimethyl sulfoxide (DMSO), 2.19 g of mPEG-SS-COOH, 0.383 g of N-(3-dimethylaminopropyl)23841

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

Research Article

ACS Applied Materials & Interfaces N′-ethylcarbodiimide hydrochloride, and 0.23 g of N-hydroxysuccinimide were activated for 3 h. One gram of H-PAMAM was also dissolved in DMSO, and the above-mentioned activated solution was slowly added dropwise under stirring and reacted at 25 °C for 1 d. Subsequently, the pH was adjusted to about 7 with a 1 M NaOH solution. The solution was lyophilized after dialysis with a 3500 Da dialysis bag. 2.5. Preparation of HG/CD@DOX. First, 0.04 g of H-PAMAM-SSmPEG and 0.008 g of DOX·HCl were dissolved in 20 mL of deionized water under stirring for 2 h. Then, after adding 3.8 μL of triethylamine, stirring was continued for another 2 h. Subsequently, the above solution was blended with 20 mL of α-CD (0.1944 g) aqueous solution and stirred for 1 d. Finally, it was lyophilized after dialysis for 1 d with the 3500 Da dialysis bag to obtain a DOX-loaded supramolecular assembly. The drug-loading content (DLC) and drug-loading efficiency (DLE) were obtained according to the following formulas DLC (wt%) =

weight of loaded drug × 100% weight of polymer

(1)

DLE (wt%) =

weight of loaded drug × 100% weight of feeding drug

(2)

Figure 2. (a) FTIR spectra of H-PAMAM, mPEG-SS-COOH, and HG. (b) 1H NMR spectrum of HG. (c) Fluorescence spectra of HG excited by 350 nm (0.1−30 mg mL−1). (d) Fluorescence photograph of different concentrations of HG under 365 nm ultraviolet light.

2.6. In Vitro DOX Release from HG/CD@DOX. The in vitro release behavior of HG/CD@DOX was studied through dialysis at 37 °C. Briefly, 2 mL of HG/CD@DOX aqueous solution was placed in 3500 Da dialysis bags, and they were immersed in four different phosphate-buffered saline (PBS) solutions (pH = 7.4, glutathione (GSH) = 0 mg mL−1; pH = 7.4, GSH = 3 mg mL−1; pH = 5, GSH = 0 mg mL−1; and pH = 5, GSH = 3 mg mL−1). The device was shaken at 100 rpm in the dark, and 2 mL of PBS medium was taken out at a predetermined time to analyze the DOX release while replenishing the same amount of fresh medium to keep the total amount of PBS medium unchanged. The amount of DOX released was achieved through the absorbance of the solution at 480 nm. 2.7. Cytotoxicity Assessment. The cytotoxicity of blank and drug-loaded supramolecular assemblies was determined through 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using MCF-7 cells. The cells were cultured in 96-well plates for 24 h in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The cell density was maintained at 3000 cells per well. Then, the medium containing the samples of different concentrations was added and cultured for another 24 h. The cells were washed twice with PBS to remove excess material after removing the medium. A total of 90 μL of DMEM and 10 μL of MTT were added and cultured for another 4 h. Finally, the crystals were dissolved by DMSO, and the absorbance was measured at 570 nm. Cell viability is defined as the ratio of the absorbance of the corresponding experimental group to that of the control group. 2.8. Cellular Uptake and Cellular Imaging. MCF-7 cells were cultured onto a glass dish for 24 h in a 5% CO2 incubator, and then the medium was removed. DOX, blank HG/CD, and DOX-loaded HG/ CD were dissolved in the medium, resulting in the final DOX concentration of 10 μg mL−1. The medium was removed after incubating the cells for 1 and 3 h using the different mixtures described above. Finally, the cells were fixed with 4% paraformaldehyde and observed through confocal laser scanning microscopy (CLSM). 2.9. Statistical Analysis. All of the experiments were performed in triplicate, and the results are presented as the average ± standard deviation.

(FTIR) and NMR spectra. The absorption peak at 1728 cm−1 was attributed to the newly formed carbonyl group in mPEG-SSCOOH (Figure 2a). As with H-PAMAM, typical absorption bands for amide I and amide II were also found in HG at 1645 and 1550 cm−1. Compared with the spectrum of H-PAMAM, HG showed new absorption peaks at 2876 and 1100 cm−1, which can be attributed to the CH3 end group and C−O in mPEG-SS-COOH, respectively. The peaks within 3500 to 3200 cm−1 were significantly decreased, indicating that most of the terminal amino groups on the surface of H-PAMAM participated in the reaction. Based on this, it can be concluded that HG was successfully synthesized. The synthesis of HG was further confirmed by 1H and 13C NMR spectra, and the chemical shifts of various protons and carbons are shown in Figures 2b and S5. Since H-PAMAM was inside HG and a large number of PEG chains were grafted around it, its peaks were not obvious in the NMR spectra. The molecular weight of HG was tested by gel permeation chromatography using dimethylformamide as the mobile phase. Its number-average molecular weight was about 18 500 Da, and the PDI was 1.11, indicating that the molecular weight distribution was very narrow. 3.2. AIE Effect of HG. As shown in Figure 2c, when water was used as solvent, HG exhibited a very strong fluorescence emission at 455 nm. In addition, the fluorescence intensity of HG continuously increased as its concentration increased, exhibiting a typical AIE effect. A similar phenomenon was observed from a photograph under 365 nm ultraviolet light (Figure 2d). Previous studies28−30 have shown that H-PAMAM has fluorescence emission, whose fluorescence source is attributed to the oxidation of tertiary amines. HG was fabricated with H-PAMAM as the core. When the concentration of HG increases, the molecules aggregate. The abundant amide groups promote the formation of hydrogen bonds, increasing the rigidity of the molecular structure, weakening the nonradiative transition of the chromophore, and enhancing the fluorescence emission. 3.3. Construction of Supramolecular Assembly HG/ CD. The polypseudorotaxane formed by PEG and α-CD is widely reported because of its relatively good solubility in

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of HG. The synthesis route of HG is shown in Figure 1a. According to the previous work,34,37 H-PAMAM and mPEG-SS-COOH were synthesized first (Figures S1−S4, Supporting Information). Then, mPEG was grafted to the surface of H-PAMAM by acylation to obtain HG with a hyperbranched structure. The structure of HG was verified by Fourier transform infrared 23842

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

Research Article

ACS Applied Materials & Interfaces

Figure 3. (a) Fluorescence spectra of HG (1 mg mL−1) and HG/CD (equivalent to 1 mg mL−1 HG) with λex = 350 nm. (b) TEM and DLS (inset) images of HG/CD.

Figure 4. (a) Fluorescence spectra of HG/CD in PBS at different pH values and (b) intensity at 445 or 450 nm. (c) Fluorescence spectra of HG/CD in aqueous solution with the addition of GSH (3 mg mL−1) and (d) intensity at 440 or 455 nm against time.

Figure 5. (a) TEM and DLS (inset) images of HG/CD@DOX. (b) Release of DOX from HG/CD@DOX with different pH values (5.0 or 7.4) and GSH concentrations (0 or 3 mg mL−1). Student’s t-test, P < 0.01.

fluorescence intensity was significantly increased after adding a certain concentration of α-CD (5 mM) to HG (Figure 3a). Combined with a previous study,40 the enhancement of fluorescence emission can be attributed to the increase in the

aqueous solution and ability to self-assemble and aggregate in solution.38,39 We mixed HG with α-CD to obtain an HG/CD supramolecular assembly. The emission efficiency of the HG/ CD assembly was tested by fluorescence spectra. The 23843

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

Research Article

ACS Applied Materials & Interfaces

Figure 6. (a) Emission spectrum of HG/CD and the absorption spectrum of DOX. (b) Fluorescence spectra of HG/CD, DOX, and HG/CD@DOX with λex = 350 or 480 nm. (c) Fluorescence lifetime of H-PAMAM in HG/CD and HG/CD@DOX. (d) Fluorescence spectra of HG/CD@DOX at varying times during drug release in PBS (pH = 5, GSH = 3 mg mL−1) with λex = 350 nm.

number of HG chains threaded with α-CD, facilitating the formation of hydrogen bonds between α-CD, increasing chain rigidity, and limiting chain motion in both linear unimers and micellar assemblies. The morphology and size distribution of the HG/CD supramolecular assembly were measured through a transmission electron microscope (TEM) and dynamic light scattering (DLS), respectively (Figure 3b). It can be seen from the TEM photograph that blank HG/CD supramolecular assemblies are spherical, having a particle diameter of about 55 nm. The size measured by DLS is 119 nm, and these changes in size are due to the two different states of the supramolecular assemblies being swollen in solution and shrunk after drying.10 3.4. pH and Redox Dual Responsiveness of Supramolecular Assembly HG/CD. HG/CD is based on HPAMAM, which exhibits pH sensitivity, and its size and structure can vary with pH.41 The fluorescence intensity of HG/CD at different pH values was tested through fluorescence spectra. With the decrease in pH, the fluorescence intensity of HG/CD increased continuously and was very sensitive to the change of pH (Figure 4a,b). On the other hand, disulfide bonds are chemical bonds with redox sensitivity, which can responsively break under reducing conditions, and are relatively stable under normal physiological conditions; however, when they enter tumor cells, they will be cleaved by overexpressed GSH.42,43 Therefore, the supramolecular assemblies containing

disulfide bonds are rapidly decomposed in the GSH solution. The fluorescence intensity of HG/CD with time after the addition of GSH was measured by fluorescence spectra (Figure 4c,d). Interestingly, as time went on, the fluorescence intensity of HG/CD increased continuously and then reached a plateau, and the emission peak position was blue-shifted. We hypothesized that after grafting the flexible chain PEG, the fluorescence emission of H-PAMAM was inhibited, when the disulfide bonds were broken, the fluorescence emission was restored. To verify our hypothesis, we added GSH to a pure HG solution and observed the change in fluorescence intensity over time. As shown in Figure S6, similar to HG/CD, the fluorescence intensity of HG also increased with time and then reached a plateau. A similar trend in both indicates that the supramolecular assembly rapidly disintegrates in the GSH environment. 3.5. Preparation and In Vitro Release of HG/CD@DOX. The anticancer drug DOX was encapsulated in HG/CD by dialysis. H-PAMAM is a three-dimensional macromolecule with high branching, abundant surface functional groups and abundant cavities. DOX can be encapsulated in the internal cavities by hydrophobic interactions.44,45 The hydrogen bond between α-CD and PEG can lock DOX in H-PAMAM, ensuring that it is not easily leaked. It can be seen from the TEM photograph that the particle size and shape of HG/CD@DOX 23844

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

Research Article

ACS Applied Materials & Interfaces

are about 73 nm and spherical, respectively (Figure 5a). The size of 155 nm measured by DLS. Such a particle size distribution is advantageous for an effective EPR effect for passive targeting of cancer cells. In addition, the DLC and DLE of HG/CD@DOX were measured to be 4.26 and 50.99%, respectively. The in vitro release behavior of HG/CD@DOX was studied in four different PBS buffer solutions (pH = 7.4, GSH = 0 mg mL−1; pH = 7.4, GSH = 3 mg mL−1; pH = 5, GSH = 0 mg mL−1; and pH = 5, GSH = 3 mg mL−1) at 37 °C. The DOX release within 48 h was less than 20% under the conditions of pH = 7.4 and GSH = 0 mg mL−1, which indicated that HG/CD@DOX can slowly diffuse under physiological conditions (Figure 5b). It is beneficial to reduce the damage of DOX to the body system. At the same pH, the addition of GSH can accelerate the release of DOX because GSH breaks the disulfide bond in the supramolecular assembly and disintegrates the assembly. Similarly, a decrease in pH can also accelerate the release of DOX. H-PAMAM itself exhibits pH sensitivity, and its size and structure can vary with pH. It has a loose structure in a weakly acidic environment, which is beneficial for releasing the loaded DOX. On the other hand, a decrease in pH can enhance the ζ-potential of HG/CD and HG/ CD@DOX, which will facilitate the cellular uptake of supramolecular drug carriers (Figure S7).19 The pH/redox dual responsiveness of the supramolecular assembly means that when HG/CD@DOX is used for cancer treatment, it can remain stable during blood circulation and selectively release rapidly at the tumor to achieve a good therapeutic effect. 3.6. FRET Effect of HG/CD@DOX. In addition to the AIE effect, the FRET effect of HG/CD@DOX was also investigated. As shown in Figure 6a, the fluorescence emission peak of HG/ CD overlaps with the UV absorption peak of DOX, indicating that there may be a FRET effect when DOX is loaded on HG/ CD. When HG/CD@DOX was excited at 350 nm, two emission peaks were observed in the fluorescence spectrum. A weak emission peak appears at around 450 nm, belonging to HG/CD, and another emission peak appears at around 600 nm, which is attributed to DOX (Figure 6b). The above results indicate that HG/CD is first excited by 350 nm, and the emission peak appears at about 450 nm. This emission peak, in turn, activates DOX as the excitation light of DOX, demonstrating the FRET effect between HG/CD and the loaded DOX. In addition, the fluorescence lifetime of H-PAMAM in HG/CD is 6.34 ns. When the drug was loaded, the lifetime of H-PAMAM in HG/CD was reduced to 6.00 ns (Figure 6c). This also indicated the presence of the FRET process in HG/CD@DOX, since the appearance of FRET is generally combined with a shortened fluorescence lifetime of the donor.35 The dynamic release process of the

Figure 7. CLSM images of MCF-7 cells after incubation with HG/CD (A), DOX (B), and HG/CD@DOX (C) for 1 or 3 h. The numbers 1, 2, 3, and 4 represent the images of bright-field, channel of H-PAMAM, DOX, and overlay, respectively (scale bar 50 μm).

Figure 8. CLSM images of MCF-7 cells after incubation with HG/CD (A) and HG/CD@DOX (B) for 3 h. The numbers 1, 2, and 3 represent the images of the channels of H-PAMAM, FRET, and DOX, respectively (scale bar 50 μm).

Figure 9. (a) Cytotoxicity of HG/CD to MCF-7 cells after incubation for 24 h. (b) Cell viabilities of MCF-7 cells after incubation with HG/CD@ DOX and DOX·HCl for 24 h. Student’s t-test, P < 0.01. 23845

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

Research Article

ACS Applied Materials & Interfaces

the FRET effect between HG and DOX. Therefore, we believe that this material will have potential applications in the area of anticancer drug delivery and monitoring.

loaded drug can be monitored by the FRET effect. During the release of DOX, the emission peak at around 600 nm (the emission peak of DOX) decreased with time, whereas the emission peak at around 425 nm (the emission peak of HPAMAM) increased (Figure 6d). This result indicates that the FRET effect between the donor and the acceptor gradually decreased as the drug was released. 3.7. Cell Uptake and Cell Imaging. Cellular uptake and cell imaging capabilities of HG/CD@DOX were investigated by CLSM using MCF-7 cells. After incubation with the cells for 1 h, the blue fluorescence of the blank and DOX-loaded HG/CD clearly appeared in the cytoplasm (Figure 7), suggesting that the supramolecular assembly could be rapidly endocytosed. In B, red fluorescence appeared in both the cytoplasm and the nucleus because of the good water solubility of DOX·HCl. After being internalized by cells, DOX·HCl could quickly reach the nucleus through a passive diffusion mechanism. Unlike B, most of the red fluorescence in C appeared in the cytoplasm, indicating that the loaded DOX was not released in large amounts. When incubated for 3 h, more samples entered the cells, resulting in the fluorescence intensity becoming stronger. The red fluorescence of DOX was clearly observed in the nucleus, while most of the blue fluorescence of HG/CD appeared in the cytoplasm. The above results indicate that the supramolecular assembly can be rapidly internalized by the cells and continuously release loaded drug with good bioimaging ability. The FRET phenomenon could also be observed in cells by acquiring the emission of HG/CD, DOX, and FRET channels through CLSM. HG/CD (donor), DOX (acceptor), and FRET channels were excited using 405, 488, and 405 nm lasers, respectively. Accordingly, fluorescence emission was collected at 410−500, 550−625, and 550−625 nm, respectively. HG/CD@ DOX emitted blue fluorescence in the donor channel and red fluorescence in the FRET and acceptor channels (Figure 8, combined with Figure 6), indicating an obvious FRET effect. 3.8. In Vitro Cell Viability Assay. The cytotoxicity of the blank HG/CD supramolecular assembly was determined through MTT assay using MCF-7 cells. When the concentration reached 2 mg mL−1, the cell viability was still above 90%, indicating that HG/CD had excellent biocompatibility (Figure 9a). When loaded with DOX, HG/CD@DOX showed a potent killing effect on MCF-7 cells (Figure 9b). Moreover, the viability of MCF-7 cells gradually decreased as the concentration of DOX increased. Compared with free DOX·HCl, HG/CD@DOX exhibited a slower endocytosis and release, which resulted in a lower anticancer activity. The above results indicate that the HG/CD supramolecular assembly had excellent biocompatibility and exhibited good tumor therapeutic ability after loading DOX.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b04938. Materials; characterizations; synthetic route of mPEG-SSCOOH; FTIR and 1H NMR spectra of H-PAMAM and mPEG-SS-COOH; 13C NMR spectrum of HG; fluorescence spectra of HG with the addition of GSH; and ζpotential of HG/CD and HG/CD@DOX at various pH values (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.L.). *E-mail: [email protected] (H.J.). *E-mail: [email protected] (C.W.). ORCID

Caiqi Wang: 0000-0002-9733-2954 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The financial support from the National Natural Science Foundation of China (51671179) is gratefully acknowledged. REFERENCES

(1) Maeda, H. Tumor-Selective Delivery of Macromolecular Drugs via the EPR Effect: Background and Future Prospects. Bioconjugate Chem. 2010, 21, 797−802. (2) Torchilin, V. Tumor Delivery of Macromolecular Drugs Based on the EPR Effect. Adv. Drug Delivery Rev. 2011, 63, 131−135. (3) Yu, M.; Zheng, J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS Nano 2015, 9, 6655−6674. (4) Duan, Q.; Cao, Y.; Li, Y.; Hu, X.; Xiao, T.; Lin, C.; Pan, Y.; Wang, L. pH-Responsive Supramolecular Vesicles Based on Water-Soluble Pillar[6]arene and Ferrocene Derivative for Drug Delivery. J. Am. Chem. Soc. 2013, 135, 10542−10549. (5) Webber, M. J.; Langer, R. Drug Delivery by Supramolecular Design. Chem. Soc. Rev. 2017, 46, 6600−6620. (6) Zhang, J.; Ma, P. X. Cyclodextrin-Based Supramolecular Systems for Drug Delivery: Recent Progress and Future Perspective. Adv. Drug Delivery Rev. 2013, 65, 1215−1233. (7) Zhou, J.; Yu, G.; Huang, F. Supramolecular Chemotherapy Based on Host-Guest Molecular Recognition: A Novel Strategy in the Battle against Cancer with a Bright Future. Chem. Soc. Rev. 2017, 46, 7021− 7053. (8) Chen, D.; Dong, X.; Qi, M.; Song, X.; Sun, J. Dual pH/Redox Responsive and CD44 Receptor Targeting Hybrid Nano-Chrysalis Based on New Oligosaccharides of Hyaluronan Conjugates. Carbohydr. Polym. 2017, 157, 1272−1280. (9) Hu, J.; Zhuang, W.; Ma, B.; Su, X.; Yu, T.; Li, G.; Hu, Y.; Wang, Y. Redox-Responsive Biomimetic Polymeric Micelle for Simultaneous Anticancer Drug Delivery and Aggregation-Induced Emission Active Imaging. Bioconjugate Chem. 2018, 29, 1897−1910. (10) Wang, X.; Yang, Y.; Zhuang, Y.; Gao, P.; Yang, F.; Shen, H.; Guo, H.; Wu, D. Fabrication of pH-Responsive Nanoparticles with an AIE Feature for Imaging Intracellular Drug Delivery. Biomacromolecules 2016, 17, 2920−2929. (11) Yang, Q.; Li, L.; Sun, W.; Zhou, Z.; Huang, Y. Dual StimuliResponsive Hybrid Polymeric Nanoparticles Self-Assembled from POSS-Based Starlike Copolymer-Drug Conjugates for Efficient Intra-

4. CONCLUSIONS In summary, we constructed a supramolecular nanodrug delivery system, HG/CD@DOX, with intrinsic AIE and FRET effects in a simple, economical, and rapid method. Through an ingenious structural design, the drug delivery system offers pH/redox dual responsiveness, which allows the drug delivery system to remain stable for an extended duration when circulating in blood and selectively release the anticancer drug at the tumor tissue. At the same time, the cytotoxicity test showed that HG/CD itself has excellent biocompatibility, as well as a high proliferation inhibition effect on MCF-7 after loading DOX. Importantly, drug delivery and dynamic release processes can be monitored using the AIE effect of HG/CD and 23846

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847

Research Article

ACS Applied Materials & Interfaces cellular Delivery of Hydrophobic Drugs. ACS Appl. Mater. Interfaces 2016, 8, 13251−13261. (12) Zhang, Y.; Guo, Z.; Cao, Z.; Zhou, W.; Zhang, Y.; Chen, Q.; Lu, Y.; Chen, X.; Guo, Q.; Li, C.; Liang, D.; Sun, T.; Jiang, C. Endogenous Albumin-Mediated Delivery of Redox-Responsive Paclitaxel-Loaded Micelles for Targeted Cancer Therapy. Biomaterials 2018, 183, 243− 257. (13) Luo, J. D.; Xie, Z. L.; Lam, J. W. Y.; Cheng, L.; Chen, H. Y.; Qiu, C. F.; Kwok, H. S.; Zhan, X. W.; Liu, Y. Q.; Zhu, D. B.; Tang, B. Z. Aggregation-Induced Emission of 1-Methyl-1,2,3,4,5-Pentaphenylsilole. Chem. Commun. 2001, 1740−1741. (14) Jiang, X.; Gao, H.; Zhang, X.; Pang, J.; Li, Y.; Li, K.; Wu, Y.; Li, S.; Zhu, J.; Wei, Y.; Jiang, L. Highly-Sensitive Optical Organic Vapor Sensor through Polymeric Swelling Induced Variation of Fluorescent Intensity. Nat. Commun. 2018, 9, No. 3799. (15) Li, M.; Gao, Y.; Yuan, Y.; Wu, Y.; Song, Z.; Tang, B. Z.; Liu, B.; Zheng, Q. C. One-Step Formulation of Targeted Aggregation-Induced Emission Dots for Image-Guided Photodynamic Therapy of Cholangiocarcinoma. ACS Nano 2017, 11, 3922−3932. (16) Li, M.; Long, S.; Kang, Y.; Guo, L.; Wang, J.; Fan, J.; Du, J.; Peng, X. De Novo Design of Phototheranostic Sensitizers Based on StructureInherent Targeting for Enhanced Cancer Ablation. J. Am. Chem. Soc. 2018, 140, 15820−15826. (17) Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Aggregation-Induced Emission: The Whole Is More Brilliant than the Parts. Adv. Mater. 2014, 26, 5429−5479. (18) 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. (19) Zhuang, W.; Xu, Y.; Li, G.; Hu, J.; Ma, B.; Yu, T.; Su, X.; Wang, Y. Redox and pH Dual-Responsive Polymeric Micelles with AggregationInduced Emission Feature for Cellular Imaging and Chemotherapy. ACS Appl. Mater. Interfaces 2018, 10, 18489−18498. (20) Yu, G.; Wu, D.; Li, Y.; Zhang, Z.; Shao, L.; Zhou, J.; Hu, Q.; Tang, G.; Huang, F. A Pillar[5]arene-Based [2]rotaxane Lights up Mitochondria. Chem. Sci. 2016, 7, 3017−3024. (21) Chen, X.; He, Z.; Kausar, F.; Chen, G.; Zhang, Y.; Yuan, W. Z. Aggregation-Induced Dual Emission and Unusual Luminescence beyond Excimer Emission of Poly(ethylene terephthalate). Macromolecules 2018, 51, 9035−9042. (22) Dou, X.; Zhou, Q.; Chen, X.; Tan, Y.; He, X.; Lu, P.; Sui, K.; Tang, B. Z.; Zhang, Y.; Yuan, W. Z. Clustering-Triggered Emission and Persistent Room Temperature Phosphorescence of Sodium Alginate. Biomacromolecules 2018, 19, 2014−2022. (23) Wang, D. J.; Imae, T. Fluorescence Emission from Dendrimers and Its pH Dependence. J. Am. Chem. Soc. 2004, 126, 13204−13205. (24) You, Y.-Z.; Yu, Z.-Q.; Cui, M.-M.; Hong, C.-Y. Preparation of Photoluminescent Nanorings with Controllable Bioreducibility and Stimuli-Responsiveness. Angew. Chem., Int. Ed. 2010, 49, 1099−1102. (25) Yuan, W. Z.; Zhang, Y. Nonconventional Macromolecular Luminogens with Aggregation-Induced Emission Characteristics. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 560−574. (26) Hu, R.; Leung, N. L. C.; Tang, B. Z. AIE Macromolecules: Syntheses, Structures and Functionalities. Chem. Soc. Rev. 2014, 43, 4494−4562. (27) Huang, T.; Wang, Z.; Qin, A.; Sun Jing, Z.; Tang Ben, Z. Luminescent Polymers Containing Unconventional Chromophores. Acta Chim. Sin. 2013, 71, 979−990. (28) Cao, L.; Yang, W.; Wang, C.; Fu, S. Synthesis and Striking Fluorescence Properties of Hyperbranched Poly(amido amine). J. Macromol. Sci., Part A: Pure Appl.Chem. 2007, 44, 417−424. (29) Yang, W.; Pan, C.-Y. Synthesis and Fluorescent Properties of Biodegradable Hyperbranched Poly(amido amine)s. Macromol. Rapid Commun. 2009, 30, 2096−2101. (30) Sun, M.; Hong, C.-Y.; Pan, C.-Y. A Unique Aliphatic Tertiary Amine Chromophore: Fluorescence, Polymer Structure, and Application in Cell Imaging. J. Am. Chem. Soc. 2012, 134, 20581−20584. (31) Hu, W.; Qiu, L.; Cheng, L.; Hu, Q.; Liu, Y.; Hu, Z.; Chen, D.; Cheng, L. Redox and pH Dual Responsive Poly(amidoamine)

Dendrimer-Poly(ethylene glycol) Conjugates for Intracellular Delivery of Doxorubicin. Acta Biomater. 2016, 36, 241−253. (32) Hu, W.; Cheng, L.; Cheng, L.; Zheng, M.; Lei, Q.; Hu, Z.; Xu, M.; Qiu, L.; Chen, D. Redox and pH-Responsive Poly(amidoamine) Dendrimer-poly(ethylene glycol) Conjugates with Disulfide Linkages for Efficient Intracellular Drug Release. Colloids Surf., B 2014, 123, 254−263. (33) Yang, W.; Pan, C.-Y.; Liu, X.-Q.; Wang, J. Multiple Functional Hyperbranched Poly(amido amine) Nanoparticles: Synthesis and Application in Cell Imaging. Biomacromolecules 2011, 12, 1523−1531. (34) Zhao, J.; Dong, Z.; Cui, H.; Jin, H.; Wang, C. Nanoengineered Peptide-Grafted Hyperbranched Polymers for Killing of Bacteria Monitored in Real Time via Intrinsic Aggregation-Induced Emission. ACS Appl. Mater. Interfaces 2018, 10, 42058−42067. (35) Han, X.; Liu, D.-E.; Wang, T.; Lu, H.; Ma, J.; Chen, Q.; Gao, H. Aggregation-Induced-Emissive Molecule Incorporated into Polymeric Nanoparticulate as FRET Donor for Observing Doxorubicin Delivery. ACS Appl. Mater. Interfaces 2015, 7, 23760−23766. (36) Yuan, Y.; Liu, B. Visualization of Drug Delivery Processes Using AIEgens. Chem. Sci. 2017, 8, 2537−2546. (37) Zhang, H.; Zhao, Y. Polymers with Dual Light-Triggered Functions of Shape Memory and Healing Using Gold Nanoparticles. ACS Appl. Mater. Interfaces 2013, 5, 13069−13075. (38) Kai, D.; Low, Z. W.; Liow, S. S.; Karim, A. A.; Ye, H.; Jin, G.; Li, K.; Loh, X. J. Development of Lignin Supramolecular Hydrogels with Mechanically Responsive and Self-Healing Properties. ACS Sustainable Chem. Eng. 2015, 3, 2160−2169. (39) Karim, A. A.; Loh, X. J. Design of a Micellized AlphaCyclodextrin Based Supramolecular Hydrogel System. Soft Matter 2015, 11, 5425−5434. (40) Liow, S. S.; Zhou, H.; Sugiarto, S.; Guo, S.; Chalasani, M. L. S.; Verma, N. K.; Xu, J.; Loh, X. J. Highly Efficient Supramolecular Aggregation-Induced Emission-Active Pseudorotaxane Luminogen for Functional Bioimaging. Biomacromolecules 2017, 18, 886−897. (41) Liu, Y.; Bryantsev, V. S.; Diallo, M. S.; Goddard, W. A., III PAMAM Dendrimers Undergo pH Responsive Conformational Changes without Swelling. J. Am. Chem. Soc. 2009, 131, 2798−2799. (42) Cheng, R.; Meng, F.; Deng, C.; Klok, H.-A.; Zhong, Z. Dual and Multi-Stimuli Responsive Polymeric Nanoparticles for Programmed Site-Specific Drug Delivery. Biomaterials 2013, 34, 3647−3657. (43) Zhao, D.; Zhang, H.; Yang, S.; He, W.; Luan, Y. Redox-Sensitive mPEG-SS-PTX/TPGS Mixed Micelles: An Efficient Drug Delivery System for Overcoming Multidrug Resistance. Int. J. Pharm. 2016, 515, 281−292. (44) Li, J.; Liang, H.; Liu, J.; Wang, Z. Poly (Amidoamine) (PAMAM) Dendrimer Mediated Delivery of Drug and pDNA/siRNA for Cancer Therapy. Int. J. Pharm. 2018, 546, 215−225. (45) Lin, L.; Fan, Y.; Gao, F.; Jin, L.; Li, D.; Sun, W.; Li, F.; Qin, P.; Shi, Q.; Shi, X.; Du, L. UTMD-Promoted Co-Delivery of Gemcitabine and miR-21 Inhibitor by Dendrimer-Entrapped Gold Nanoparticles for Pancreatic Cancer Therapy. Theranostics 2018, 8, 1923−1939.

23847

DOI: 10.1021/acsami.9b04938 ACS Appl. Mater. Interfaces 2019, 11, 23840−23847