A glutathione-activated carrier-free nanodrug of triptolide as a trackable drug delivery system for monitoring and improving tumor therapy

Ying Li ab, Lihua Zhou bc, Baode Zhu d, Jingjing Xiang b, Jian Du e, Manwen He c, Xingxing Fan f, Pengfei Zhang *b, Ruosheng Zeng *ag and Ping Gong *b
aSchool of Materials Science and Engineering, School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin 541004, P. R. China
bGuangdong Key Laboratory of Nanomedicine, Shenzhen Engineering Laboratory of Nanomedicine and Nanoformulations, CAS Key Lab for Health Informatics, CAS-HK Joint Lab for Biomaterials, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences Shenzhen 518055, China. E-mail: pf.zhang@siat.ac.cn; ping.gong@siat.ac.cn
cSchool of Applied Biology, Shenzhen Institute of Technology, No. 1 Jiangjunmao, Shenzhen 518116, P. R. China
dCollege of Chemistry Biology & and Environmental Engineering, Xiangnan University, Chenzhou, 423043, China
eDepartment of Urology, The First Affiliated Hospital of Shandong First Medical University, No. 16766, Jingshi Road, Jinan 250000, P. R. China
fState Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau, SAR, China
gSchool of Physical Science and Technology, Guangxi University, Nanning 530004, China. E-mail: zengrsh@guet.edu.cn

Received 16th March 2021 , Accepted 17th May 2021

First published on 18th May 2021


Abstract

Triptolide (TP) is one of the most common systemic treatments for inflammatory and immune diseases in China for centuries. However, TP exhibits some disadvantages, such as poor solubility in water, poor bioavailability, liver toxicity, renal toxicity, and other side effects. In order to reduce the adverse effects of TP, researchers have developed numerous strategies to address the adverse properties of triptolide. Nano-carrier-based triptolide delivery systems represent an emerging technology and are one of the strategies of nanomedicine that combines diagnostic and therapeutic applications in a single agent. In this approach, we developed a glutathione-activated carrier-free nanodrug of triptolide (CyssTPN) as a trackable drug delivery system. In this system, CyssTP self-assemble to form a carrier-free nanodrug, which possesses a monodisperse spherical morphology with hydrodynamic average sizes of about 50 nm. In addition, CyssTPN had good stability under different physiological conditions (pH, high salt, etc.). Apart from cellular imaging and cell uptake, CyssTPN can be tracked by the activation of TP ability in real-time and applied for cancer cell treatment efficiently. The result showed that CyssTPN could improve solubility, reduce the side effects, and increase the bioavailability of triptolide. It could also track triptolide activation timely and tumor therapy successfully.


Introduction

Triptolide (TP),1 a structurally unique diterpenoid, was isolated from the traditional Chinese medicinal plant Thunder God Vine (Tripterygium wilfordii Hook. f., TwHf), which belongs to genus Tripterygium, Celastraceae family. It has been used as an immunosuppressive and anti-inflammatory medicine for centuries.2–4 Besides, triptolide has been shown to be an effective anti-cancer drug to treat a wide range of cancers.5–7 Although triptolide has attracted extensive attention from researchers due to its excellent efficacy in treating numerous diseases,8 its clinical development over the past two decades has been limited by liver toxicity,9 renal toxicity,10 and other side effects,11 along with poor solubility and poor bioavailability of triptolide.12 Therefore, it is urgent to improve the solubility of triptolide, reduce side effects, and increase bioavailability to enhance the therapeutic efficacy and accelerate its clinical applications.13–15

Based on the literature, researchers have developed various strategies to address the adverse properties of triptolide. One of the strategies is the nano-carrier-based triptolide delivery systems.16–19 Up to now, a variety of nanoparticles, including liposomes,20,21 vesicles,22 and DNA nanoparticles,15 loaded with triptolide were reported.23 With these nanoplatforms, the pharmacokinetics and biodistribution of triptolide could be changed, resulting in improved therapeutic efficiency and reduced side effects. However, carrier-based triptolide delivery systems always suffered from low triptolide loading ratio, batch-to-batch variation in triptolide drug loading, and long-term toxicity of carriers, which greatly hinder their rapid clinical translation.24

In order to overcome the disadvantages of carrier-based delivery methods, carrier-free nanodrugs based on small molecules have been developed.24–26 Noncarrier nanodrugs have a high and precise drug loading ratio and negligible nanotoxicity, which exhibit nanoscale characteristics to realize intracellular delivery by themselves without the aid of additional nanocarriers.27–29 Besides, compared with traditional drug delivery platforms, most carrier-free nanodrugs consisting of pure drugs, drug–drug dimers, drug derivatives could conduct drug delivery without any additional nanocarriers, which are just excipients without direct therapeutic function.30–32 Thus, the drug loading capacities of carrier-free nanodrugs are excellent, and some of them even reach up to 100%.33,34 Besides, the drug loading ratio of carrier-free nanodrugs is precise and tunable because of the well-defined structure of small molecules.35 There is no concern with carrier-induced longterm toxicity for avoiding the use of additional carriers.36

In this study, we synthesized a glutathione-activated carrier-free nanodrug of triptolide (CyssTPN) to improve the solubility of triptolide, reducing side effects and increasing bioavailability. The nanodrug was constructed by conjugating Cy (IR780) to triptolide (TP) via a disulfide bond. The newly synthesized small molecules of CyssTP self-assemble to form carrier-free nanodrugs in an aqueous environment (Fig. 1). It is well known that the level of glutathione (GSH) is unusually high in the tumor microenvironment,37–39 and CyssTPN could be activated by the high levels of glutathione in the tumor.40,41 The experimental results show that the nanodrugs apparently improved the solubility of triptolide and hugely reduced the toxicity of free TP. Furthermore, owing to intramolecular charge transfer between Cy and TP, the structure of Cy in nanodrugs showed an absorption peak at 654 nm in UV-vis spectroscopy. However, when the disulfide bond of the nanodrug was broken by glutathione, a new absorption peak at 787 nm of Cy was observed. Correspondingly, the fluorescence (FL) emission peak at 757 nm (excitation at 646 nm) turned to 800 nm (excitation at 745 nm). Through a series of experimental verification, the nanodrug has been successfully utilized for the real-time tracking of the TP activation and tumor therapy. Our studies further encourage the applications of carrier-free nanodrugs and real-time tracking of activation of the drug.


image file: d1qm00400j-f1.tif
Fig. 1 Schematic of the chemical structure of CyssTP and its self-assembly into a glutathione-activated carrier-free nanodrug of triptolide (CyssTPN) as a trackable drug delivery system for tumor therapy.

Results and discussion

It is known that heptamethine cyanine dyes have drawn extensive attention in recent years because of their improved photophysical properties and readily modified chemical structures, among which indocyanine green (ICG) as a NIR heptamethine cyanine dye has been approved by the US Food and Drug Administration (FDA) for clinical diagnosis.42,43 IR780 (Cy) is also one of the heptamethine cyanine probes that has been widely studied in the field of cancer theranostic.44–48 However, very few reports have been provided about carrier-free nanodrugs based on heptamethine cyanine dyes.49,50 In this study, we synthesized a structurally similar carrier-free nanodrug of triptolide (CyssTPN and CyccTPN). They were synthesized using the same connection linkages (disulfide bond, –S–S–) on one end to replace the chlorine atom in the meso-position of Cy and on the other end to condense with TP-COOH (Fig. S1, ESI). Structural validations of the related synthesis are demonstrated using 1H NMR and 13C NMR spectroscopy techniques and high-resolution mass spectrometry (HRMS) in the ESI (Fig. S2–S6).

The as-synthesized CyssTPN show spherical morphologies with an average diameter of around 50 nm as obtained via transmission electron microscopy (TEM) and dynamic laser scattering (DLS) techniques, respectively (Fig. 2a). Similarly, CyccTPN also presents spherical morphologies with a diameter of around 60 nm, as shown in the transmission electron microscopy (TEM) and dynamic laser scattering (DLS) images in Fig. S7 (ESI). We characterized its responsiveness towards GSH by DLS, and the results showed that the CyssTPN presents different particle sizes under the condition of different concentrations of GSH. When the concentration of GSH reached a specific limit (160 μM), they were decomposed into small molecules (Fig. S8, ESI). We expected that carrier-free nanodrugs could enhance tumor accumulation and reduce side effects compared to free therapeutic drugs. In addition, compared to TP, the solubility of CyssTPN significantly increased, as shown in Fig. S9 (ESI). It could effectively improve the biocompatibility, which was useful for further in vitro investigation. Next, the UV-vis spectra of nanodrugs were recorded. The results showed that when Cy is covalently bonded to TP, and the absorption peak of Cy is blue-shifted from 787 nm to 654 nm. That is probably due to the intramolecular charge transfer between Cy and TP (electronic stretching group), resulting in a shift of 133 nm (Fig. 2b). Then, with the increase in GSH, the absorption of CyssTPN at 787 nm gradually increased, while the absorption at 654 nm decreased gradually (Fig. 2b). More importantly, the absorption ratio (787 nm/654 nm) of nanodrugs had a linear relationship with GSH concentration (Fig. S10a, ESI). The ultraviolet-visible absorption spectrum of CyccTPN has no similar results (Fig. S10b and Fig. S11a, ESI).


image file: d1qm00400j-f2.tif
Fig. 2 Particle size and TEM image of CyssTPN (a), nanodrug size: 50 nm. (b) UV-Vis absorption changes of CyssTPN (100 μM) in the presence of GSH (0–250 μM) in DMSO/PBS (1/1, v/v, PH = 7.4). Fluorescence spectra responses of CyssTPN (100 μM) to GSH (0–250 μM) (λex = 646 nm/λem = 757 nm (c), λex = 745 nm/λem = 800 nm (d)).

The fluorescence spectrum of CyssTPN was studied subsequently. Two different excitation wavelengths (646 nm and 745 nm) were chosen to excite CyssTPN. The nanodrugs showed a strong fluorescence emission peak at 757 nm (λex = 646 nm) and a weak fluorescence emission peak at 800 nm (λex = 745 nm). Then, with the increase in the concentration of GSH, the fluorescence intensity of the Cy group in the nanodrugs at 757 nm (λex = 646 nm) gradually decreased, while the fluorescence intensity at 800 nm (λex = 745 nm) increased (Fig. 2c and d). On the contrary, with different concentrations of GSH, the fluorescence signal of CyccTPN was almost unchanged (Fig. S11b, ESI). Therefore, the significant changes in the fluorescence spectrum of the carrier-free CyssTPN during the reaction with GSH could monitor the activation of CyssTPN. The reaction kinetics of the nanodrugs and glutathione was studied. The fluorescence emission intensity of CyssTPN at 800 nm (λex = 745 nm) increased within 30 min, while CyccTPN showed negligible changes. To further verify whether CyssTPN can release the active drug triptolide (TP), we conducted HPLC monitoring, as depicted in Fig. S12 (ESI), to show the drug release process of interactions between CyssTPN and GSH. Moreover, we performed corresponding studies on the stability with respect to pH, high-salt, and photostability of CyssTPN, as shown in Fig. S13, S14, and S15 (ESI). The pH, high concentration of Na+, and the effect of light on the CyssTPN size had negligible influence, which shows that CyssTPN can be used under different conditions.

Density functional theory (Gaussian09 B3LYP) calculations were further used to interpret the photophysical properties of Cy, CyccTP, and CyssTP. Based on their geometrically optimized structures from DFT, the calculated molecular orbitals and energy levels showed that the energy gaps of Cy, CyccTP, and CyssTP are 2.023, 2.354, and 2.273 eV, respectively (Fig. 3). The highest occupied molecular orbital (HOMO) orbital was well delocalized on the donor unit and acceptor unit, while the lowest unoccupied molecular orbital (LUMO) orbital was more distributed on the electron-deficient acceptor, indicating that ICT was along the molecular skeleton. It should be noted that the energy gap for LUMO–HOMO of Cy was substantially less than the LUMO–HOMO of CyssTP, which was consistent with longer wavelength absorption for Cy, as compared to CyssTP and CyccTP.


image file: d1qm00400j-f3.tif
Fig. 3 Density functional theory (DFT) optimized structures and frontier molecular orbitals (MOs) of Cy (a), CyccTP (b), and CyssTP (c) in DMSO. Calculations were based on the ground state geometry by DFT at the B3LYP/6-31G (d) level using Gaussian 09.

In addition, it can be clearly seen from the optimized structure that CyssTP molecules tend to form a strongly twisted conformation along the main skeleton, with a dihedral angle above 19.55°, and the dihedral angle of CyccTP was 23.59°. On the contrary, the torsional movement of Cy was more suppressed, and the dihedral angle was 3.97°, indicating effective planar conjugation (Fig. 4). In brief, the experimental and theoretical values were approximately consistent.


image file: d1qm00400j-f4.tif
Fig. 4 Optimized structures with the dihedral angles along the molecular backbones, from top-view and side-view, for Cy (a), CyccTP (b), and CyssTP (c).

Based on the excellent fluorescence absorption properties of CyssTPN, we used laser scanning confocal microscopy (CLSM) and flow cytometry to monitor the cellular uptake efficacy of the nanodrugs. CyssTPN and CyccTPN were co-cultured with cells. After 30 min, the cells were stained using Mito-tracker. The results showed that the MCF-7 cancer cells exhibited light red fluorescence of nanodrugs. Also, it is indicated that MCF-7 cancer cells exhibited advantageous uptake efficiency to CyssTPN and CyccTPN. Besides, the result also revealed that the green signals of Mito-tracker (excited by 488 nm) were mainly overlapped with the red ones exhibited by CyssTPN/CyccTPN, which indicated that CyssTPN was preferentially located in the mitochondria (Fig. 5a). Mitochondria have been long recognized as an ideal subcellular target for cancer therapy. Therefore, nanodrugs present mitochondrial targeting effects, which may be more beneficial to improve the efficiency of tumor therapy. We also added MCF-7 cells with CyssTPN, cultured for 30 min, and imaged at 10/15/20/25/30 min. It is indicated that MCF-7 cancer cells exhibited advantageous uptake efficiency to CyssTPN (Fig. S16, ESI)


image file: d1qm00400j-f5.tif
Fig. 5 Fluorescence confocal microscope images of MCF-7 cancer cells cultured with CyssTPN/CyccTPN (5 μM) for 30 min, respectively. Scale bar: 20 μm. Time-dependent flow cytometry analysis of the cellular uptake of CyssTPN (b) real-time tracking of the fluorescence intensity of the drug release. Cancer cells: MCF-7.

To further certify the TP release ability of CyssTPN/CyccTPN in cells, the flow cytometry experiment was carried out in GSH high level MCF-7 cells and GSH low level L02 cells. We added CyssTPN and CyccTPN to MCF-7/L02 cells, respectively, recorded the fluorescence intensity of the cell uptake and drug release in point-in-time. The experimental results showed that the fluorescence intensity of CyssTPN decreased at the channel of 675 nm and increased at 780 nm (Fig. 5b). However, CyccTPN had little change in different channels (Fig. S17, ESI). This is consistent with the result of Fig. 2c and d. In the control group (GSH low level L02 cells), the fluorescence intensity of CyssTPN and CyccTPN hardly changed over time (Fig. S18, ESI). The results further showed the excellent real-time tracking activation of the TP ability of CyssTPN in cancer cells.

To investigate the anti-tumor efficiency of CyssTPN, cancer cells (MCF-7) were used in this experiment. We evaluated the cytotoxicity of Cy, TP, CyccTPN, and CyssTPN. With the increase in each concentration, free TP and CyssTPN had obvious killing effects on MCF-7 (Fig. 6). On the contrary, CyccTPN exhibited poor anti-tumor activity and lower toxicity towards cancer cells. Because of the carbon–carbon covalent bond between Cy and TP, TP was not released. The results further indicated that CyssTPN could reduce significant side effects compared to free TP drugs. In addition, it might effectively improve the biocompatibility and therapy in cells due to its unique response to the tumor microenvironment and excellent nanodrug properties.


image file: d1qm00400j-f6.tif
Fig. 6 In vitro cell viability of MCF-7 cells treated with different concentrations of TP, CyccTPN, and CyssTPN, as measured using the CCK-8 kit.

Encouraged by the promising in vitro cytotoxicity and drug delivery of CyssTPN, we further investigated the nanodrugs for therapy on tumor spheroids (Fig. 7a). The tumor spheroids were constructed, to which CyccTPN and CyssTPN were added, respectively, and then the morphological changes of tumor spheroids over time were observed. The results showed that CyssTPN had satisfactory permeability of nanodrugs in tumor spheroids. Moreover, the tumor spheroids in the presence of CyssTPN had obvious morphological changes. With the change in time, the tumor spheroids kept shrinking (Fig. 7b). Simultaneously, the tumor spheroids with CyccTPN had almost no morphological changes (Fig. 7c). This result indicated that CyssTPN could be activated in tumor spheroids, and surprisingly, exhibited TP treatment effects. Finally, to further prove the tumor inhibition effects of CyssTPNin vivo, the images of hematoxylin and eosin (H&E) stained tumor slices confirmed that the therapy group using CyssTPN led to the most significant tumor tissue damage (Fig. S19, ESI), while tumor tissue in the other control groups partly or largely retained their normal morphology (Fig. S19, ESI).


image file: d1qm00400j-f7.tif
Fig. 7 Schematic of the CyssTPN drug release (a). When CyssTPN were endocytosed into the tumor spheroids, they are activated under the high level of GSH, the disulfide bond is broken, and TP is released to achieve the therapeutic effect of the tumor. Time-dependent confocal microscopy images of CyssTPN (b) and CyccTPN (c) on tumor spheroids. Scale bar: 20 μm.

Inspired and encouraged by the tumor spheroids therapy, we monitored the activation of nanodrugs CyssTPNin vivo, and the tumor-bearing mice were injected with CyssTPN. The signals of FL imaging excited by 740 nm laser were recorded at 0.5 h, 12 h, 36 h, and 72 h (Fig. S20, ESI). In tumor FL imaging, we could obtain the NIR fluorescence signals of CyssTPN excited by 740 nm after post-injection, and the fluorescence signals of CyssTPN gradually increased with the extension of time. This is in line with our expectations. The result indicated that CyssTPN had the ability of targeting the tumor. Then, the structure of the disulfide linkage in CyssTPN could be broken by high GSH levels in tumor tissue, which activated the TP.

To further investigate the in vivo nanodrug toxicity towards other organs after treatment, H&E stained images of sliced major organs collected from different groups were obtained. No noticeable abnormalities were observed in the major organs, including the liver, spleen, kidney, heart, and lungs, from the treated mice, suggesting negligible toxic side effects of carrier-free nanodrugs in vivo. However, the original drug triptolide showed strong hepatorenal toxicity (Fig. 8). The in vivo biosafety of TP, CyccTPN, and CyssTPN was investigated by liver/kidney function index, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE). As shown in Fig. 9, the concentration of ALT/AST/BUN/CRE with the TP treated mouse group showed a significant difference compared to the control group, but the nanodrugs treated mouse group showed no obvious change compared to the control group. In a word, the results of these experiments show that CyssTPN could reduce the side effects and increase the bioavailability of triptolide in vivo.


image file: d1qm00400j-f8.tif
Fig. 8 Histopathological analysis by hematoxylin and eosin (H&E) staining of heart, liver, spleen, lungs, and kidney sections isolated from nude mice after treatment with control, TP, CyccTPN, and CyssTPN, respectively. Scale bar: 100 μm.

image file: d1qm00400j-f9.tif
Fig. 9 Nude mouse serum levels of ALT, AST, BUN, and CRE after intravenous injection of different contents of TP, CyccTPN, and CyssTPN. Error bars are standard error of the mean (#P > 0.05) as compared to control.

Conclusions

In summary, we have developed a glutathione-activated carrier-free nanodrug of triptolide (CyssTPN) as a trackable drug delivery system for monitoring and improving tumor therapy. In this system, the carrier-free nanodrugs with triptolide could be activated by the high levels of glutathione in the tumor. The nanodrug apparently improved the solubility of triptolide, hugely reduced toxicity of free TP, and increased bioavailability. Moreover, the nanodrug has been successfully utilized for real-time tracking TP activation and tumor therapy. Therefore, our study indicated that CyssTPN improved the solubility of triptolide, reduced side effects, increased bioavailability, and improved its therapeutic efficacy to the benefit of accelerating its clinical applications. Last but not least, through the establishment of a carrier-free nanodrug system, the controlled release of the drug, monitoring, and intervention could be effectively integrated to achieve intelligent, precise, and efficient tumor therapy.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81671758 and 32000982), the Guangdong Natural Science Fund (2019A1515110222, 2020B1111540001), Shenzhen Science and Technology Program (JCYJ20200109114616534), the China Postdoctoral Science Foundation (2019M660219), the Chinese Academy of Sciences (Y959101001), the UNSW-CAS Collaborative Research Seed Fund Program (172644KYSB20190059) and SIAT Innovation Program for Excellent Young Researchers (201920), the Guangxi Natural Science Foundation (Grant No. 2017GXNSFGA198005), the Research Program of Shenzhen Institute of Technology (2111015). We thank the help from the office of Research and Development of Shenzhen Institute of Technology (SIT). We also acknowledge Prof. Kai Li from Southern University of Science and Technology (China) and Prof. Safacan Kolemen from the Koc University (Turkish) for their help on the density functional theory calculation and analysis.

Notes and references

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/d1qm00400j
These authors contributed equally to this work.

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