Biodegradable, Hydrogen Peroxide, and Glutathione Dual

Communication Cite This: J. Am. Chem. Soc. 2018, 140, 7373−7376

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Biodegradable, Hydrogen Peroxide, and Glutathione Dual Responsive Nanoparticles for Potential Programmable Paclitaxel Release Daiqin Chen,†,‡,§ Guoqiang Zhang,†,‡ Ruimin Li,†,‡ Mirong Guan,†,‡ Xueyun Wang,†,‡ Toujun Zou,†,‡ Ying Zhang,†,‡ Chunru Wang,† Chunying Shu,*,† Hao Hong,*,§ and Li-Jun Wan*,†,‡

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†

Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, and Beijing National Laboratory for Molecular Sciences, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § Department of Radiology, Center for Molecular Imaging, University of Michigan, Ann Arbor, Michigan 48109-2200, United States S Supporting Information *

sulfides,21,22 thioethers,23,24 thioketals,25,26 selenium,27,28 tellurium,29,30 proline oligomers,31,32 etc. Meanwhile, most GSH responsive nano-DDSs contain disulfide groups.33,34 However, only a few of these redox potential responsive nano-DDSs can simultaneously respond to ROS and GSH, which compromise the therapeutic efficacy. Consequently, researchers are enthusiastic in developing ROS and GSH dual responsive nanoDDSs to improve their drug delivery performance.35,36 Although great progress has been made, there are still some limitations that need to be overcome, such as the poor sensitivity to ROS and GSH simultaneously at their biological concentrations, the suboptimized biocompatibility and biodegradability of the nano-DDSs, the promiscuous fate of byproducts after fulfilling their tasks, and so on.37−40 Herein, ROS and GSH dual responsive TKN was synthesized, which could respond to H2O2 and GSH at the biological level, producing the byproducts of biocompatible acetone and lipoic acid (Figure S1). The drug release and cell studies results validated that PTX-TKNs could specifically release PTX in the presence of H2O2 and GSH, suggesting that this nano-DDS might fulfill the programmable on-demand drug release both extracellularly and intracellularly in cancer cells (Scheme 1). The in vivo antitumor efficacy of PTX-TKNs on PC-3 tumor bearing mice showed that PTX-TKNs could not only significantly inhibit the growth of PC-3 tumors but also greatly alleviate the side effects of PTX and improve the quality of life thereof. To guarantee the biocompatibility of the subsequently biodegraded byproducts, we chose dihydrolipoic acid (DHLA) as the monomer, which is the reductive product of lipoic acid (LA), a FDA-approved antioxidant.41,42 The presence of the two −SH proton peaks at 1.28 and 1.32 ppm in the 1H NMR spectrum of DHLA verified the successful reduction of lipoic acid (Figure S2). The ROS responsive oligomer (o-DHLA) was synthesized through a condensation reaction of DHLA and 2,2-dimethylpropane (DMP). The 1H NMR spectrum clearly demonstrated the presence of a thioketal group corresponding to the proton peak at 1.58

ABSTRACT: Reactive oxygen species (ROS) and glutathione (GSH) dual responsive nanoparticulate drug delivery systems (nano-DDSs) hold great promise to improve the therapeutic efficacy and alleviate the side effects of chemo drugs in cancer theranosis. Herein, hydrogen peroxide (H2O2) and GSH dual responsive thioketal nanoparticle (TKN) was rationally designed for paclitaxel (PTX) delivery. Compared to other stimulisensitive nano-DDSs, this dual responsive DDS is not only sensitive to biologically relevant H2O2 and GSH for ondemand drug release but also biodegradable into biocompatible byproducts after fulfilling its delivering task. Considering the heterogeneous redox potential gradient, the PTX loaded TKNs (PTX-TKNs) might first respond to the extracellular ROS and then to the intracellular GSH, achieving a programmable release of PTX at the tumor site. The selective toxicity of PTXTKNs to tumor cells with high levels of ROS and GSH was verified both in vitro and in vivo.

T

he past decades have witnessed the tremendous development of nano-DDSs for cancer chemotherapy.1−3 A concept has been widely accepted that ideal nano-DDSs should be able to not only keep the payloads from any passive leakage in the delivery process but also readily unload the cargoes in a controlled release manner once arriving at the target sites.4−6 In order to meet these criteria, researchers have proposed many stimuli-responsive nano-DDSs for the targeted delivery and site-specific release of various therapeutic cargos.7−9 Both external (including light, magnetic field, ultrasound, etc.) and internal stimuli (e.g., pH, temperature, enzyme, redox potential, etc.) have been intensively utilized to trigger the smart nanoDDSs.10−13 Among these reported triggers, redox potential has attracted much attention in the development of nano-DDSs for cancer theranosis due to the uniquely heterogeneous redox potential gradient in the tumor site.14−16 The extracellular matrix is oxidative as a result of overproduction of ROS, while the intracellular cytoplasma is reductive because of a relative high level of GSH in most tumor sites.17,18 There are many ROS-sensitive moieties, including boronic esters/acids,19,20 © 2018 American Chemical Society

Received: November 13, 2017 Published: May 25, 2018 7373

DOI: 10.1021/jacs.7b12025 J. Am. Chem. Soc. 2018, 140, 7373−7376

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Journal of the American Chemical Society

delivery. The disappearance of the proton peaks of −SH in oDHLA suggested the formation of disulfide bonds after the exposure to oxygen (Figure 1d). The cross-linked product was characterized with gel permeation chromatography (GPC), with MW of 41 290 (Figure S3). The drug loading content (DLC) was determined by high performance liquid chromatography (HPLC) to be 10.2% (Figures S4 and S5). The transmission electron microscopy (TEM) images demonstrated that the PTX-TKNs was ca. 250 nm (Figure 2a), which agreed well with the dynamic light scattering (DLS)

Scheme 1. Schematic Illustration of the ROS and GSH Dual Responsive Nano-DDSa

a

The structure of the ROS and GSH dual responsive nano-DDSs, containing both ROS responsive (purple) and GSH responsive (green) motifs.

ppm (Figure 1a). Notably, there were still obvious proton peaks at 1.28−1.32 ppm in o-DHLA (marked by red double stars in

Figure 2. TEM images and size distribution of PTX-TKNs response to H2O2 (a,b) and r-GSH (c,d), respectively. Scale bars in (a), inset in (a), and (c) represent 500, 100, and 100 nm, respectively. NR release (e) and dual-staged NR release (f) triggered with 100 mM H2O2 and 10 mM r-GSH. Figure 1. (a) 1H NMR spectrum of o-DHLA (red star-marked peak corresponds to the impurities). (b) MALDI-TOF spectrometry of oDHLA (red star-marked peak corresponds to the matrix rather than oDHLA). (c) 1H NMR spectra of o-DHLA, o-DHLA treated with 100 mM H2O2, lipoic acid, and acetone in CDCl3. (d) 1H NMR spectra of o-DHLA before and after exposure to oxygen flow in CDCl3.

results (Figure 2b). Both TEM images and size distribution results showed that PTX-TKNs degraded into ca. 40 nm of smaller nanoparticles after incubation with 100 mM H2O2 (Figure 2a,b). The ζ-potential changes also confirmed the presence of degradation products after H2O2 treatment, as a characteristic peak appeared in the negatively charged region (Figures S6 and S7), and the population of PTX-TKNs declined while the amount of their degradation byproducts (40 nm particles) increased with the elevation of H2O2 concentration (Figures S7−S9). Notably, the PTX-TKNs were sensitive to H2O2 as low as 100 μM (Figures S8 and S9), a biologically relevant level of H2O2 (50−100 μM). GSH treatment only led to slight swelling of PTX-TKNs, with the diameter changing from 250 to 260 nm and the constant ζpotential of PTX-TKNs (Figures 2c,d and S10). The NR release curve showed that only 21% drug release was observed under the normal condition; while the drug release reached up to 62% and 87% after treatment with GSH or H2O2 within 24 h, respectively (Figure 2e). The dual-stage responsive NR release performance showed that the passive NR release almost reached plateau within 4 h, then the NR release was greatly accelerated when H2O2 was added, and a further expedition of

Figure 1a), indicating the existence of active sulfhydryl residues. The matrix-assisted laser desorption/ionization-time-of-flight mass (MALDI-TOF) spectrometry indicated that the oligomer contains 15 units of DHLA (Figure 1b). As expected, the low polymerization degree preserved active sulfhydryl residues and facilitated the subsequent cross-linking to incorporate GSH responsiveness in the final product. After treatment with H2O2, an obvious signal was observed at 2.17 ppm corresponding to acetone, a product of the oligomer cleavage (Figure 1c). The other product, LA, was confirmed by the appearance of peaks around 1.72 ppm. All the above results demonstrated that the obtained oligomer was biodegradable, and most importantly, its degradation products (lipoic acid and acetone) were quite biocompatible and safe for clinical uses.43,44 The oligomer was then cross-linked to form disulfide bonds and used for PTX 7374

DOI: 10.1021/jacs.7b12025 J. Am. Chem. Soc. 2018, 140, 7373−7376

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to PC-3 cells or GSH-elevated CHO cells (Figures S15 and S16). All the observations indicated that PTX-TKNs posed much higher selective cytotoxicity to PC-3 and GSH-elevated CHO cells than that to normal CHO cells, most probably resulting from the ROS and GSH triggered drug release from PTX-TKNs. Finally, we tested the anticancer efficacy of PTX-TKNs on PC-3 tumor bearing mice. PTX-TKNs showed obvious accumulation at the tumor site after intravenous injection at 24 h postinjection (Figure S17). The tumor size chart and H&E slices clearly showed that both Taxol (free PTX) and PTX-TKNs could effectively inhibit tumor growth, with the inhibition rate of 77% and 65% on the 20th day, respectively (Figure 4b,c). The body weight fluctuation (Figure 4a), the

NR release was observed once the addition of GSH at 3 h postaddition (Figures 2f, S11, and S12). All these results confirmed that this nano-DDS could respond to H2O2 and GSH, triggering on-demand drug release correspondingly. The ROS responsiveness of PTX-TKNs was evaluated on PC-3 cells with an inherently higher level of ROS, and CHO cells were selected as the control (Figure S13). After treatment of 6.9 μM free PTX, the cell viabilities of PC-3 cells and CHO cells fell to 63% and 4%, respectively, suggesting that free PTX did more harm to CHO cells than to PC-3 cells (Figure 3a).

Figure 4. Body weight fluctuation curves (a), tumor growth charts (b), tumor H&E slices (c). Scale bar = 100 μm. Alanine aminotransferase (ALT, d) and aspartate aminotransferase (AST, e) levels of mice injected with saline, Taxol, and PTX-TKNs, respectively (n = 5); **p < 0.01. Figure 3. Cell viabilities of PC-3 cells and CHO cells incubated with free PTX (a), TKNs (b), and PTX-TKNs (c) at a series of gradient concentrations. (d) Cell viabilities of CHO cells and CHO cells pretreated with GSH-OEt incubated with PTX-TKNs at a series of gradient concentrations. n = 6; *p < 0.05, **p < 0.01. (e) Microscopic images of CHO and PC-3 cells treated with TKNs (1 mg/mL) or PTX-TKNs (13.8 μM, with respect to PTX). Scale bar = 20 μm.

alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of the mice treated with Taxol suggested obvious side effects of free PTX to the mice; however, the mice treated with PTX-TKNs did not show any abnormality. Considering the selective cytotoxicity of PTX-TKNs to ROS-rich PC-3 cells and GSH-elevated CHO cells rather than to normal cells, which was verified previously in the in vitro assays, all these results should probably be ascribed to the on-demand drug release triggered from PTX-TKNs by high levels of ROS and GSH in the heterogeneous redox potential tumor environment. In conclusion, a H2O2 and GSH dual-responsive DDS was designed for PTX effective delivery. This dual responsive property could lead to on-demand cargo release in tumor microenvironment due to the higher level of H2O2 and GSH, which has been verified both in vitro and in vivo. This nano DDS platform is biocompatible and biodegradable, with the byproducts being lipoic acid and acetone. Moreover, other chemotherapeutic agents or genes might also be selectively delivered by this smart dual responsive carrier. Therefore, the

While after incubating with PTX-TKNs (13.8 μM), the cell viabilities of PC-3 cells and CHO cells were 30% and 90%, respectively. No significant cell death was observed in these two cells after treatment with TKNs (Figure 3b), as confirmed by the microscopy images in Figure 3e. Then some CHO cells were treated with glutathione reduced ethyl ester (GSH-OEt) to elevate the intracellular GSH level, and GSH-OEt treatment did negligible harm to CHO cells (Figure S14). After treatment with 13.8 μM PTX-TKNs, the cell viabilities of the pretreated and control CHO cells were 57% and 91%, respectively (Figure 3d). On the contrary, neither the non-ROS responsive nor the non-GSH responsive nanoparticles posed selective cytotoxicity 7375

DOI: 10.1021/jacs.7b12025 J. Am. Chem. Soc. 2018, 140, 7373−7376

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proposed drug delivery system would represent an attractive platform to enhance the bioavailability and reduce the side effects of chemotherapy drugs.

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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/jacs.7b12025. Experimental details (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] *[email protected] ORCID

Chunru Wang: 0000-0001-7984-6639 Hao Hong: 0000-0002-9730-9367 Li-Jun Wan: 0000-0002-0656-0936 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 51672280) and the Chinese Academy of Sciences (XDA09030302).

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DOI: 10.1021/jacs.7b12025 J. Am. Chem. Soc. 2018, 140, 7373−7376