Nano-Drug Design Based On The Physiological Properties Of Glutathione Ⅱ

Nano-Drug with -SMono thioether bond (-S-) as a binder is widely applied in anti-tumor and nano-drug delivery system design. Cong et al. [73] successfully developed a novel dual redox-responsive prodrug nanosystem (PTX-S-OA/TPGS NPs) assembled by hydrophobic small-molecule prodrugs. PTX-S-OA/TPGS NPs were signifificantly superior to disulfifide conjugate (PTX-2S-OA) in terms of dual redox-sensitive drug release and in vivo antitumor efficacy. PTX-S-OA/TPGS NPs have an impressive high drug loading and are effective in selectively releasing drugs at the tumor site, as shown in Figure 5A. Meng et al. [74] synthesized a new prodrug DTX-S-LA, which utilized mono thioether bond as a linker to bridge linoleic acid (LA) and docetaxel (DTX). DTX-S-LA self-assembled with DEPEG-PEG to form nanoparticles with a drug loading capacity of 53.4%. These nanoparticles had the characteristics of uniform particle size, high blood stability, and fast drug release in tumor cells and had higher tumor inhibition rates in vivo compared with free DTX, as shown in Figure 5B. Zhang et al. [75] synthesized a kind of CUR-S-CUR prodrug by coupling two CUR molecules with mono-thioether bonds for GSH-responsive drug delivery, as shown in Figure 5C. These CUR-S-CUR NPs exhibited good colloid stability, more efficient cellular uptake, and intracellular/nuclear drug delivery compared to free CUR. 

Figure 5. Schematic design of different GSH-responsive anticancer drugs with -S-. (A) Schematic representation of the preparation of PEGylated prodrug NPs of PTX-S-OA and cleavage by GSH or ROS [73]; (B) schematic representation of DTX-S-LA self-assemble in water and cleavage with GSH in tumor cells [61]; (C) schematic representation of CUR-S-CUR prodrug self-assemble and its uptake by tumor cells [75]. 

Nano-Drug with Pt-O

Pt-O bond can be reduced and cleaved by GSH to release active metabolite Pt(II). Based on this theory, Ling et al. [76] designed the GSH-sensitive prodrug nanoparticles Pt(IV) for effective drug delivery and cancer therapy. Pt(IV) nano-drugs could resist thiol-mediated detoxification through GSH depletion. After Pt(IV) nanoparticles are reduced by GSH, Pt-O broke down and released enough active Pt(II) metabolites, which covalently bonded with the target DNA and induced apoptosis (Figure 6A). Huang et al. [77] found that Pt(IV)NP-cRGD exhibited strong echogenic signals and excellent echo persistence under ultrasound imaging. Furthermore, the GSH-sensitive drug delivery system not only maximized the therapeutic effect but also reduced the toxicity of chemotherapy. Pt(IV)NP-cRGD, together with ultrasound imaging, depleted GSH, and increased ROS levels, leading to mitochondria-mediated apoptosis (Figure 6B). 

Figure 6. Self-assembled Pt (IV) nanoparticles for specifific delivery of Pt drugs. (A) Pt (IV) was reduced with GSH to Pt (II) [76]. (B) Pt(IV)NP-cRGD was reduced with GSH to Pt (II) [77]. 

Nano-Drug with Se-Se Diselenide-conjugated bond (Se-Se) has a unique dual redox sensitivity. High expression of GSH in tumors or ROS generation by oxidative stress, such as H2O2, can break the diselenide-conjugated bond to complete the redox response. Manjare et al. [78] synthesized a new GSH reduction-triggered flfluorescent probe (A) by connecting two molecules of BODIPY-Se by the diselenide-conjugated bond, which could be utilized to detect GSH or H2O2 in cancer cells. Diselenide-conjugated bond of the flfluorescent probe (A) was cleaved by GSH, then reacted with ROS to emit fluorescence. Han et al. [79] prepared the flfluorescent molecule diselenide SeDSA nanoparticles containing 9, 10-distyrylanthracene (DSA) derivative (SeDSA) with aggregation-induced emission (AIE). SeDSA could co-assemble with the antitumor prodrug and diselenide-containing paclitaxel (SePTX), to form SeDSA-SePTX Co-NPs (Co-NPs). SeDSA-SePTX Co-NPs rapidly disintegrate and release AIE dye and PTX under the reducing environment, which played the role of tumor imaging and tumor therapy. Zhao et al. [80] designed diselenide-crosslinked polymer gels (SeSey-PAA-TPEx) via free radical copolymerization. The diselenide crosslinker in the gels could be fragmented in the presence of H2O2 or GSH due to its redox-responsive property for the diagnosis of a tumor.

Click Here To Get Cistanche Herbs For Anti-Cancer

Nano-Drug with Se-N

The conjugate bond of Se-N is a novel dual redox-sensitive bond, which is not only responsive with GSH to form Se-H, but also responsive with H2O2 to form Se-N, achieving dual redox responsive effect. Xu et al. [81] developed a new dual redox-sensitive flfluorescent probe (Cy-O-Eb) based on this theory, which could dynamically track the changes of H2O2 and GSH in living cells, and directly monitored the redox status of cells. The apoptosis process of the HepG2 tumor was successfully observed by Cy-O-Eb. In this report, the breakage and generation of Se-N bond in the structure cause a change in fluorescence in the flfluorescent probe under two different environments. Under the action of GSH, Se-N bond breaks and generates Se-H structure, and the fluorescence intensity is greatly reduced. On the contrary, the Se-N bond was regenerated and the fluorescence was restored under the effect of H2O2, as shown in Figure 7.

Figure 7. The dual reaction of probe (Cy-O-Eb) with GSH/H2O2 [81]. The Se-N bond (strong fluorescence) in Cy-O-Eb was reduced with GSH to form Se-H bond (weak fluorescence). Se-N was regenerated and the fluorescence was restored under the effect of H2O2 .

Nano-Drug with -SeMono selenium bond (-Se-) is an oxidation stimuli responsive bond, which is mainly oxidized by ROS, such as H2O2, and is ruptured to release nano-drugs. Wang et al. [82] prepared the drug-loaded polymeric nanoparticles of selenium-inserted copolymer (I/DSe-NPs). I/D-Se-NPs rapidly dissociate in a few minutes mediated by ROS and promoted the continuous release of anti-tumor drugs. Moreover, Jiang et al. [83] developed a dual stimuli-responsive and wormlike micelle system (C11-Se-C11) using a switchable selenium-containing surfactant. Zhang et al. [84] designed a viscoelastic wormlike micellar solution based on a new redox-responsive surfactant, namely sodium dodecylselanylpropyl sulfate (SDSePS). The above selenium bond in nanoparticles can be oxidized by H2O2 to form Se=O to play relative activity.

5.3.4. Glutathione Responsive Photodynamic Therapy

Phototherapy can be divided into photothermal therapy (PTT) and photodynamic therapy (PDT). PTT is a treatment method for killing tumors by injecting photothermal materials into the body and irradiating them with near-infrared light (750~1400 nm). When tumor tissues/cells are heated to 40–45 ◦C, cell membranes and nucleic acids are damaged or mitochondrial dysfunction occurs in the process of hyperthermia. Prolonged exposure to high heat eventually leads to the death of tumor tissue/cells. During PTT, tumor tissue/cells have a lower heat tolerance than normal tissue/cells. Therefore, it is possible to selectively kill tumor tissues/cells by using the ability of local tumor heating, while not harming the normal tissues/cells [85].

PDT has emerged as a technique for disease treatment that requires three essential components: photosensitizers (PS), specifific wavelengths of light (ultraviolet light, visible and near-infrared light), and oxygen. Light excitation at a specifific site triggers a photochemical reaction in PS resulting in the production of reactive oxygen species (ROS), which subsequently yields tissue/cells damage and death. PDT can provide an accurate stimulus that triggers ROS production at a defined time and specifific site resulting in a signifificant reduction of off-target effects on healthy tissues [86,87].

The concentration of intracellular ROS directly determines the effect of photodynamic therapy. Hence, a decline in GSH can increase the level of ROS and promote cell apoptosis, which provides the primary theory for photodynamic therapy. Ruan et al. [88] constructed a nanosystem, Cu-tryptone nanoparticles (Cu-Try NPs), that promoted photodynamic therapy through the consumption of GSH. It demonstrated that Cu-Try NPs could deplete GSH to increase intracellular ROS and improve photodynamic therapy. Chen et al. [89] developed a kind of hydrophobic cysteine-based polydisulfifide amide (Cys-PDSA) polymers and utilized them as black phosphorus quantum dots nanocarrier. Paclitaxel (PTX) was loaded into the nanoparticles to achieve a combination of chemotherapy and photothermal therapy for cancer through GSH reduction mediated by the disulfifide bond. Yang et al. [90] prepared a new type of pH/GSH multi-response chitosan nanoparticles (SA-CS-NAC), and SA-CSNAC-loaded photosensitizer ICG to form the amphoteric mercapto chitosan nanoparticles (SA-CS-NAC@ICG NPs) by self-assembly. SA-CS-NAC@ICG NPS successfully achieved multi-response to release ICG in a microenvironment with low pH and high GSH in tumor cells. At the same time, in vitro cell experiments confirmed that SA-CS-NAC@ICG NPS had strong cell uptake ability, low biotoxicity, and good tumor inhibition. 

6. Nano-Drug Design Based on the Role of GSH in Neurological Diseases 

GSH takes participated in the neurodegenerative changes of Parkinson’s disease, mainly against the production of intracellular ROS during oxidative stress. The concentration of GSH in the substantia nigra in patients with Parkinson’s disease dramatically decreased, indicating a close relationship between GSH, oxidative stress, and Parkinson’s disease. Based on the theory above, Ma et al. [91] prepared Ag44(SR)30 silver nanoclusters with a ligand of 5-mercapto-2-nitrobenzoic acid and completed the high precision detection of GSH, which enables more accurate and comprehensive diagnosis and assessment of Parkinson’s disease. It had been reported that autism spectrum disorders (ASD) were also associated with GSH [92–95]. The research found that both reduced GSH and total GSH levels were lower in the ASD group than that in the control group [96]. In addition, some studies had found that the treatment with GSH could effectively protect renal tubular epithelial cells, reduce the occurrence of acute renal damage or even acute renal failure, and improve the survival rate of patients with cerebral hemorrhage [97]. Although GSH is directly or indirectly involved in the pathogenesis of neurological diseases, nano-drug design based on the role of GSH in oxidative stress has not been reported. This is a weakness and blind area in nanoscience research, we can make full use of the advantages of nanotechnology, combining the characteristics of nervous system diseases to develop new targeted nano-drugs. 

7. Fluorescent Nano-Probe Design Based on Physiological Properties of GSH 

The traditional methods for the visual quantitative determination of intracellular ROS and GSH are mostly instrumental analyses. However, the sample pre-treatment process is complicated, the determination is time-consuming, and the GSH and ROS in vivo cannot be monitored in real-time. In contrast, flfluorescent probe technology has the advantages of high sensitivity, good selectivity, and good real-time performance, which show outstanding features for monitoring GSH and ROS in vivo and in vitro [98–100]. The following is an introduction to the design of flfluorescent nano-probes based on the physiological properties of GSH, hoping to provide some references for the clinical application of nano-probes through the summary of this paper. 

Liu et al. [101] synthesized a novel two-photon fluorescence probe MT-1 for the detection of biological mercaptans mainly GSH in mitochondria. 4-dinitrobenzene sulfonyl group (DNBS) in a fluorescent probe, which acted as the responsive group of GSH. The Fluorescence of the probe would be quenched due to the electron-absorbing action of DNBS. But when the probe reacted with GSH in mitochondria, DNBS was eliminated, and the fluorescence of the probe was restored to directly observe biological mercaptan in living cells and tissues, which were used to detect and observe cell status. Chen et al. [102] prepared a flfluorescent probe for the detection of GSH in aqueous solution and living cells by introducing dinitrophenyl ether into 2-(20 -hydroxy-30 -ethoxyphenyl) benzothiazole. The fluorescence of the probe was quenched due to the strong electron-absorbing of the nitro group, but when the probe was reduced by GSH, the fluorophore was released to emit a strong fluorescence at 485 nm. Both of the above designs introduce a strong electron-absorbing group into the probe structure, and the fluorescence of probe is quenched or resurrected after GSH regulation. There are also some references for the application of this design [103–109]. 

All the above are small molecular flfluorescent probes, and their poor tumor-targeted ability and solubility have limited their application in vivo. In order to effectively penetrate tumors, especially those tumors with dense stroma, Niko et al. [110] designed a GSHresponsive flfluorescent probe in which the amphiphilic flfluorescent material NR12D was self-assembled and coated with a polymer DSP containing disulfifide bonds. Li et al. [111] prepared micelles by covalently linking the NIR flfluorescent dye dimethyl-4H-pyran (DCM) with the anti-tumor drug gemcitabine using a disulfifide bond as a bridge to achieve the targeted positioning and therapeutic effect of the nanoprobe. Zhang et al. [112] synthesized a GSH-responsive probe using the flfluorescent material amantadine-naphthalimide and the anticancer drug camptothecin to achieve active fluorescence imaging in cancer cells. Lu et al. [113] used hollow mesoporous carbon (HMC) coated with doxorubicin and grafted reduction-sensitive near-infrared dye (HMC SS-CDPEI) to prepare a nanoprobe to monitor the release of doxorubicin. Choi et al. [114] designed and synthesized a GSH-responsive flfluorescent carbon nanoprobe. All these probes disintegrate under the action of GSH, and the fluorescence emission can monitor the drug release in real-time.

8. Nano-Imaging Design Based on Physiological Properties of GSH 

Nano-imaging technology is to design GSH-responsive nanoparticles in which nano-imaging materials are encapsulated in the nanoparticles for dual-mode imaging and combination therapy. Li et al. [115] reported that the drug paclitaxel (PTX) and hydroxyethyl starch were bonded by disulfifide bonds, and then the fluorophore DiR was encapsulated in the nanoparticle nucleus during self-assembly, during which the DiR fluorescence was quenched. When the nanoparticles were endocytosed by tumor cells, the disulfifide bonds were cleaved by excessive GSH, resulting in the simultaneous release of DiR and PTX in the nanoparticles. The fluorescence of DiR recovered and could be applied in photoacoustic imaging. Yang et al. [116] synthesized a GSH-responsive hyaluronic acid (HA) and poly (ε-caprolactone) copolymer nanoparticle encapsulated with DOX and superparamagnetic iron oxide (SPIO). Under the action of high levels of GSH, disulfifide bonds of these nanoparticles broke, releasing internal DOX and SPIO. SPIO could be utilized in magnetic resonance imaging, while DOX was used in chemotherapy, allowing the combination of imaging and chemotherapy. Yang et al. [117] reported that amphiphilic dextran derivatives were developed from disulfide-linked dextran-g-poly-(N-3-carbobenzyloxy-L-lysine) graft polymer (Dex-g-SS-PZLL) and used as theranostic nanocarriers for chemotherapy and magnetic resonance imaging. Consequently, these reduction-sensitive nanoparticles are promising theranostic nanocarriers for magnetic resonance imaging and chemotherapy.

9. Application of Nanoscale GSH in the Food Field 

The design of sodium alginate and chitosan bilayer-modified GSH nanoliposomes was reported by Wei et al. [118]. The results of storage stability and gastrointestinal stability showed that sodium alginate and chitosan bilayer-modified liposomes not only enhanced the stability of GSH but signifificantly reduced the release rate of GSH in the gastrointestinal tract. Therefore, in a complex food-processing system, the use of sodium alginate and chitosan bilayer-modified liposomes could avoid the rapid release of GSH, increase the stability of GSH, and thus promote the absorption of GSH by gastrointestinal cells, and enhance the nutritional value of food. This study provides a reference basis and data support for the application of GSH nanoliposomes modified by sodium alginate and chitosan in food fifield.

10. Summary and Perspectives

GSH tablets and GSH injections are widely used in clinics. GSH is a kind of polypeptide, which does not exist stably during transportation and preservation, which brings some difficulties for clinical preservation, transportation, and application. Therefore, it is very important to develop nano-drugs and technologies based on the pathological characteristics of GSH so that GSH can play a much greater role in clinical practice. However, GSH nanoparticles are limited to basic experiments and have not been widely used in clinical practice. In view of the problems faced by nanotechnology in clinical diseases, it is necessary to design intelligent nanoparticles with the help of interdisciplinary integration. Nanoparticles adjust their chemical and biological functions by stimulating responsive structural changes, so as to realize intelligent biomedical applications, which is a new interdisciplinary research direction.

of GSH and develop excellent nano-drug design methods, which provide important scientific significance and application value for the research of related diseases that GSH participates in. 

Funding: The authors thank to the Harbin Medical University Daqing Campus Yu Weihan Outstanding Youth Fund (DQYWH201603) and Heilongjiang Province ordinary undergraduate youth innovative personnel training program (UNPYSCT-2015036). National Natural Science Foundation of China (82173153).

Conflicts of Interest: The authors declare no conflict of interest.

References 

1. Liu, Y.; Hyde, A.S.; Simpson, M.A.; Barycki, J.J. Emerging Regulatory Paradigms in Glutathione Metabolism. Adv. Cancer Res. 2014, 122, 69–101. 

2. Harington, C.R.; Mead, T.H. Synthesis of Glutathione. Biochem. J. 1935, 29, 1602–1611. [CrossRef]

3. Penninckx, M.J.; Elskens, M.T. Metabolism and Functions of Glutathione in Micro-Organisms. Adv. Microb. Physiol. 1993, 34, 239–301.

4. Bachhawat, A.K.; Yadav, S. The Glutathione Cycle: Glutathione Metabolism Beyond the Gamma-Glutamyl Cycle. IUBMB Life 2018, 70, 585–592. [CrossRef]

5. Bachhawat, A.K.; Kaur, A. Glutathione Degradation. Antioxid. Redox. Signal. 2017, 27, 1200–1216. [CrossRef] [PubMed] 

6. Jana, A.; Joseph, M.M.; Munan, S.; Sharma, K.; Maiti, K.K.; Samanta, A. A Single Benzene Fluorescent Probe for Efficient Formaldehyde Sensing in Living Cells Using Glutathione as an Amplifier. J. Photochem. Photobiol. B 2021, 214, 112091. [CrossRef] [PubMed] 

7. Shuhua, X.; Ziyou, L.; Ling, Y.; Fei, W.; Sun, G. A Role of Fluoride on Free Radical Generation and Oxidative Stress in BV-2 Microglia Cells. Mediat. Inflflamm. 2012, 2012, 102954. [CrossRef] [PubMed] 

8. Meister, A. Glutathione, Ascorbate, and Cellular Protection. Cancer Res. 1994, 54, 1969–1975. 

9. Rodrigues, C.; Percival, S.S. Immunomodulatory Effects of Glutathione, Garlic Derivatives, and Hydrogen Sulfide. Nutrients 2019, 11, 295. [CrossRef] 

10. Song, D.; Lin, Z.; Yuan, Y.; Qian, G.; Li, C.; Bao, Y. DPEP1 Balance GSH Involve in Cadmium Stress Response in Blood Clam Tegillarca granola. Front. Physiol. 2018, 9, 964. [CrossRef] [PubMed] 

11. Agarwal, P. Assessment of Anti-Aging Efficacy of the Master Antioxidant Glutathione. Int. J. Sci. Basic Appl. Res. 2017, 33, 257–265.