Redox-responsive self-assembly PEG nanoparticle enhanced triptolide for efficient antitumor treatment

Chemotherapy induces tumor cell death by directly damaging DNA or hindering cell mitosis. Some of the drawbacks of most chemotherapy are lack of target selectivity to tumor cells, and adverse drug reaction, which limit the treatment intensity and frequency. Herein, we synthesized the prodrug of triptolide (TP) coupled to vitamin E (VE) using dithiodiglycolic acid and co-dissolved with PEG2000- linoleic acid (MPEG200-LD) in ethanol. The PEGylated TP prodrug self-assembly nanoparticles (PTPPSN) were prepared via nanoprecipitation method. Besides, characterization, stability and in vitro release of the PEGylated nanometer prodrug were investigated. Furthermore, in vitro and in vivo antitumor efficacy of PTPPSN explored showed that the cytotoxicity of triptolide was significantly reduced in vitro preparation. However, in vitro and in vivo antitumor effect of PTPPSN was significantly improved compared to the original triptolide. In summary, the PEGylated nanoparticle successfully encapsulated triptolide yielded suitable cell microenvironment, and nanotechnology-related achievements. This study, therefore, provides a new method for antitumor research as well as an innovative technology for clinical treatment of malignant tumor.

A malignant tumor is a grave threat to human life, and its morbidity and mortality are rapidly increasing on yearly basis. The utilization of small-molecule compounds as chemotherapeutic agents can directly damage DNA 1 or hinder cell mitosis 2 , and induce cell death 3,4 . However, drawbacks such as lack of chemotherapeutic selectivity which causes toxicity to healthy cells 5 , alongside adverse drug reaction 6,7 , limit the treatment intensity and frequency of administration of such agents. Chemotherapy does not only damage the function of heart, liver, lung, kidney, bone marrow and other vital organs but also destroy the immune system 8 , resulting in the loss of the body's self-protection barrier to the tumor 9 . Additionally, chemotherapy aggravates tumor cell genome instability 10 , which in turn causes the tumor cells to adapt to chemotherapeutic drugs rapidly 11,12 . Therefore, the side reactions of chemotherapy and tumor resistance have become major obstacles to the treatment of cancer patients 13,14 .
Triptolide (generally known as TP or TL), a specie of diterpene lactone epoxide compound, is extracted from traditional Chinese medicinal plant Tripterygium wilfordii Hook. F. Triptolide is considered as one of the main active ingredients in the plant 15 . Reports on treatment of multiple cancers using triptolide showed that cell proliferation inhibition and cell cycle arrest of the drug is dependent on time and doses [16][17][18] . During triptolide therapy, LC3-α expression level increases 19,20 , PI3K -Akt -mTOR pathway is inhibited 21,22 , while ERK1/2 is activated, but autophagy of cell death is inducted. Due to its double-edged effect on efficacy and toxicity, the treatment dose is closer to the toxic dose. Therefore, the safety of triptolide is of great health concern to the users. Based on in-depth investigation on the structure-activity relationship of triptolide, some efficient and less toxic derivatives of triptolide have been synthesized [23][24][25][26] . The structural modification of triptolide has partly resolved its high toxicity and poor solubility. However, clinical research of triptolide derivatives are stagnated in phase I trials, a development that has reduced subsequent research and reports on the drug. We, therefore, speculated that the high toxicity of triptolide alongside low selectivity to diseased cells, while normal cells could account for the retarded clinical study. Presently, the application of triptolide has not been improved, hence, additional research on the structural modification and targeted delivery of triptolide need to be entrenched.
The broad application of nanotechnology in the field of drug delivery has played an active role in designing and building new and efficient conveyance system, which has significantly enriched drug delivery strategies and the development of biomaterials. In the field of anticancer drug delivery, for instance, nano drug carrier has unique advantages such as improving the drug effect 27 ; prolonging drug duration in blood circulation 28 ; passive targeting to the tumor site via EPR effect 29 ; improving uptake of drugs with active targeted modification by tumor cells 30 ; as well as controlling drug release in the cellular targeted area 31 . Although, there has been great progress in nanotechnology delivery system, some setbacks including low efficiency, poor stability, toxic side effects of carrier materials, crystallization and leakage of drugs during storage, still hamper their clinical applications.
As compared with normal cells, the microenvironment of tumor cells is oxidatively stressed due to the excessive joint production of glutathione (GSH) and reactive oxygen (ROS) in tumor cells 32 . This particular ROS microenvironment has been widely used in the design of REDOX stimulation responsive drug delivery systems for anticancer drugs delivery. For example, disulfide bonds have been broadly explored in developing and restoring sensitive prodrug and nano drug delivery systems 33 . The disulfide bond of TP prodrug can be rapidly fracted to reveal the free mercapto under the action of GSH, while the acyl bond is rapidly hydrolyzed. Also, many studies have shown that tumor microenvironment exists with REDOX heterogeneity, namely different tumors may produce varied levels of glutathione and reactive oxygen species [34][35][36] . Furthermore, different regions of the same tumor might also possess the REDOX heterogeneity of cell microenvironment, and this could be observed in the diverse growth stages of the tumor.
In this paper, with the bifunctional chelating agent of dithiodiglycolic acid, a precursor drug was formed with vitamin E and triptolide (TP-S-S-VE), and co-dissolved with MPEG200-LD in alcohol. The PEGylated TP prodrug self-assembly nanoparticles (PTPPSN) were prepared via nanoprecipitation method. Besides, characterization of the nanometer prodrug, stability and in vitro release were also investigated. Furthermore, the antitumor efficacy of the nano prodrug was explored, and the results showed that the cytotoxicity of triptolide was significantly reduced in vitro, while the in vitro and in vivo antitumor effect of PTPPSN was significantly improved compared to the free triptolide. The sketch of this issue can be seen in Fig. 1.

Synthesis, purification, and characterization of triptolide prodrug. All the experiments protocols
were approved by the principles of laboratory animal care and legislation in force in Zhengzhou University. The synthesis of triptolide prodrug (0.2 g) was obtained by reacting the corresponding dithiodiglycolic acids with vitamin E (VE), followed by grafting of triptolide. Accordingly, the required amount of acid was added to acetic anhydride and stirred for 2 h at 30 °C. Afterward, toluene was added to the reactive system, prior to total removal of toluene and dithiodiglycolic acid with a rotary evaporator. The residual oily liquid was dissolved in 2 mL of anhydrous dichloromethane. Subsequently, 0.1 g of VE and 4-dimethyl aminopyridine (DMAP) were added and stirred at room temperature. The mixed solution was purified using silica gel column chromatography and subsequently washed with n-hexane/ethyl acetate/acetic acid (10/1/1) to obtain VE-S-SCOOH (114 mg). Triptolide (100 mg) and VE-S-SCOOH (120 mg) were dissolved in 6 mL anhydrous dichloromethane, and added to dicyclohexylcarbodiimide (DCC, 48 mg) alongside DMAP (30 mg), and was stirred for 2 h at room temperature. Afterwards, the solution was filtered to remove dicyclohexylurea (DCU) and further purified via silica gel column chromatography with an eluent of petroleum ether/ethyl acetate anhydride (2/3) to collect the TP prodrug (80 mg). The target product was characterized using 1 H-NMR. Synthesis, purification, and characterization of mPEG2000-LD. Linoleic acid (500 mg), mPEG2000 (1000 mg), DCC (400 mg) and DMAP (240 mg) were dissolved in 20 mL anhydrous dichloromethane and stirred for 12 h. The solution was filtered to remove DCU, washed with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The organic layer was filtered, rotary evaporated to dryness and washed thrice with petroleum ether. The insoluble substance was filtered and was characterized as mPEG2000-LD by 1

H-NMR.
PEGylating TP prodrug nanoparticle preparation. One-step nano sedimentation method was applied to prepare the nanoparticles 37 . Different ratios of mPEG2000-LD were added to a fixed portion of triptolide The characterization of PTPPSN. The different proportions of self-assembled nanoparticles were characterized by particle size, zeta potential, polydispersity index, drug-loading capacity, and encapsulation rate indices. The DLS, TEM, and HPLC were adopted to investigate the effect of factors such as varying ratios of TP prodrug and mPEG2000-LD as well as temperature. Detailed parameters investigated included particle size and zeta potential detected by DLS (where the nanoparticles were diluted with deionized water to a concentration of 1 mg/ml prior determination at 25 °C using Zetasizer Nano ZS, Malvern Instruments); morphology observed with TEM; and the structure measured via 1 H-NMR. The HPLC was performed at 25 °C using a C18 column (150 mm × 4.6 mm, 5 μm, Waters, USA) and methanol-water (42:58, v/v) as mobile phase which flowed at a rate of 1 mL/min. The detection wavelength was 218 nm. Encapsulation efficiency (EE) and drug loading (DL) of the nanoparticles were calculated according to equations (1)   Cell proliferation assay. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to test cytotoxicity of TP prodrug self-assembly nanoparticle and cell viability. Briefly, 2 × 10 5 of H 22 BALB/C mouse liver cancer cell (H 22 ) per well were incubated in 200 μL medium containing 10% FBS in 96-well plates for 24 h. The cells were then exposed to series of TP, TP prodrug, TPPSN, and PTPPSN for 72 h. After drug exposition, the medium was removed and 100 μL of MTT solution (0.5 mg/mL in DMEM containing 10% FBS) was added to each well. The plates were incubated for 2 h at 37 °C and 20 μL DMSO solution was then added to each well for 4 h at 37 °C. Absorbance was measured at 570 nm using a plate reader (Biotech America). The percentage of surviving cells was calculated as the absorbance ratio of treated to untreated cells. The inhibitory concentration 50% (IC50) of the treatments was determined from the dose-response curve. All experiments were set up in quadruplicate to determine means and SDs.
In vivo antitumor study designs. All  Tissue distribution. The tumor model mice were grouped and injected intravenously through the lateral tail vein with either TP, TP prodrug, TPPSN, or PTPPSN. After intravenous injection, blood, heart, liver, spleen, lung, kidney and tumor were taken from the mice at different times (0.25, 0.5, 1, 2, 3, and 4 h). The blood sample was drawn into a 2 mL tube containing 10 μL of sodium heparin and was centrifuged at 3000 × g for 10 min to obtain the plasma, before storage at −20 °C pending further determination. Each organ was precisely taken and added to 0.1 g/mL normal saline. After crushing with high-speed shears, the samples were kept and stored in the tubes at −20 °C for HPLC detection. A 150 μL of plasma or 300 μL of the organ homogenate was added to 10 μL internal standard, prednisolone (4 μg/mL) dissolved in 1 mL of methanol. All the mixtures were vortexed for 5 min and added to 2 ml of ethyl acetate, while vortexing for 10 min. The vortexed solution was centrifuged at 8000 × g for 10 min to obtain the organic layer. The upper organic layer was dried using nitrogen blown under 40 °C water bath. The dried samples were resolved with 100 μL of the mobile phase, and centrifuged at 8000 × g for 10 min to obtain supernatant for HPLC determination.
The HPLC was performed at 25 °C using a C18 column (150 mm × 4.6 mm, 5 μm, Waters, USA), while methanol-2 mM ammonium acetate (42:48, v/v) were used as mobile phases at a flow rate of 1 mL/min. The detective wavelength was 218 nm.

Results
Synthesis of TP produg and mPEG2000-LD. The bioconjugate, TP prodrug, was obtained through the reaction of dihydroxyacetic acid (Fig. 1A). The target product, TP prodrug was dried and tested using 1 3 H, t). The result showed that mPEG2000-LD was successfully synthesized (Fig. 2B).
Synthesis, morphological characterization and physicochemical properties of self-assembly nanoparticles. PTPPSN was prepared via different ratios of mPEG2000 nanoparticles, and the particle size and zeta potential were characterized by SEM and dynamic light scattering. The results as shown in Table 1 indicate that with an increase in mPEG2000-LD concentration, the particle size of the PTPPSN also decreased, with a steady rise in zeta potential.
As shown in the TEM images in Fig. 3, TP prodrug self-assembly nanoparticles were spherical in shape and monodispersed.
Drug loading capacity and encapsulation efficiency. Drug loading capacity (DL) and encapsulation efficiency (EE) play critical roles in drug delivery. To obtain high drug loading to meet therapeutic needs, the optimized formulation was used to prepare TP prodrug self-assembly nanoparticle. The DL of the prepared PTPPSN was 57.0 ± 4.7%, along with an EE of 81.8 ± 2.8%.
Scientific REPoRtS | (2018) 8:12968 | DOI:10.1038/s41598-018-29692-0 Stability test. As shown in Fig. 4, the particle size of TPPSN increased gradually in the medium with the extension of time. However, when the mPEG -LD/TP -S -S -VE ratio was greater than 0.2, the particle size had no obvious change. The results showed the PEGylation can improve the stability of the nanoparticles.
In vitro drug release profile. Cumulative drug release from PTPPSN, TPPSN and TP were studied in different concentrations of GSH (1 μM, 10 μM, 1 mM, and 10 mM) at pH 7.4. As shown in Fig. 5A, the cumulative release rate of PTPPSN was released fastest in 1 mM of GSH at 24 h; The cumulative release of TP was 5.29%, 15.63%, 62.75% and 22.54% at 24 h in different concentration of GSH (1 μM, 10 μM, 1 mM, and 10 mM), respectively. The degradation rate of PTPPSN at 24 h was 60.56%, 29.66%, 8.39%, and 1.01%, respectively (Fig. 5B). The release study at different pH level (6.8 and 7.4) were shown in S1.
In vitro efficacy studies. The in vitro cytotoxic activity of PTPPSN against human cancer lines MCF-7 was evaluated in comparison with TP, TP prodrug, and controlled PBS. The cell viability was also verified using MTT assay. The results, presented in Fig. 6, show the concentrations required to inhibit cell growth by 50% (IC50 values). Thus, the antitumor efficacy of the self-assembled nanoparticle constructed with this prodrug has been further investigated in mice bearing an H 22 solid tumor.
In vivo antitumor efficacy. The antitumor efficacy of the PTPPSN was investigated on the mice H 22 solid tumor-burdened model, in comparison with TP, TP prodrug, and TPPSN. After tumors had reached approximately 1 cm 3 , the animals were subjected to different treatments using injection protocols, as explained in Section  Tissue distribution. TP and PTPPSN organ distribution results were shown in Fig. 8, the kinetic study of nanoparticles was shown in S2, and the HPLC spectra of TP with retention time can be found in S3. After PEGylating TP prodrug self-assembly nanoparticles (PTPPSN) preparation, TP distributed mainly in the liver, spleen and lung, especially the liver. After 1 h of administration, the distribution of PTPPSN was significantly higher in the mice tumor than TP. This was consistent with previous reports which suggested that nanoparticles are easily identified by mononuclear phagocytic system and are enriched in mononuclear macrophages of liver, spleen and lung 38 , and the

Discussion
Triptolide is pharmacologically potent, but its diverse toxic effects on organs such as liver, kidney, skin, gastrointestinal tract as well as the cardiac and reproductive system have restricted its therapeutic window 39 . Herein, we tried to reduce the toxicity of triptolide while enhancing its anti-tumor suppression activity. The PTPPSN was found to be a highly efficient low toxic tumor therapy hence could solved the systemic toxicity of triptolide. This could be due to PEGylating of the prodrug which prolonged release in plasma and Redox-responsive targeted at the tumor part.
With the development of nanotechnology, more nanomaterials are employed in drug formulations and delivery, viz., polymer micelle, liposomes, dendritic macromolecules, microemulsion, nano gold and other metal nanoparticles 40 . Nanomaterials can play both active and passive roles to target tumor cells. Due to the wide gap between the walls of the tumor tissue and incomplete lymphatic flow, nanoparticles of 10-200 nm can be passively gathered in the microenvironment of tumor tissue. This phenomenon is known as the enhanced permeability and retention (EPR) effect. However, due to different EPR effects on various tissues, the passive targeting strategy of  nanomaterials usually fails to achieve the desired target result. Active targeting refers to nanoparticles modification or connection with monoclonal antibody marked tumor cells or peptides, which can combine with the tumor cell surface receptors or specific antibodies. This study, therefore, coupled VE alongside triptolide and PEGylated to form a self-assembly of nanoparticles. The morphological observations of PTPPSN showed a uniform dispersion of spherical objects, with the size regularity being consistent with the results of particle size distribution (Fig. 3). By increasing the concentrations of mPEG2000-LD, PTPPSN became significantly smaller, while its stability also increased remarkably ( Table 1).
The cumulation rate of PTPPSN in low GSH concentration (1 μM) was lower than that of high concentration (10 mM). The in vivo environment of plasma GSH concentration was between 1 μM-10 mM, while the concentration of GSH in tumor environment ranged between 1 mM-10 mM (Fig. 5). Hence, the slow release of PTPPSN within the plasma environment enriched the tumor tissues by EPR effect. The enriched PTPPSN in tumor was quickly released by Redox reaction. This indicated that PTPPSN owned a certain tumor targeting property. The solid tumor volume of TP, TPPSN and PTPPSN groups were significantly lower than the blank control group.  Meanwhile, the inhibition rate of the solid tumor of PTPPSN was significantly higher than that of TP and TPPSN groups (Fig. 7). The results of tumor biopsy showed that TP, TP prodrug and PTPPSN could significantly inhibit tumor growth. The PTPPSN and TP were mainly distributed in organs like liver, spleen and lung but maximally in the liver (Fig. 8). The distribution results suggested that PTPPSN can effectively circumvent the mononuclear scavenger system identification through the cumulative EPR effect on tumor site.
In conclusion, this study demonstrated that more efficient diterpene lactones based anticancer nanomedicines could be designed by conjugating with VE, thereby potentiating their "target recognition" of the tumor microenvironment.