The photothermal imaging studies were performed when tumor size reached about 400 mm3 . The mice were intravenously (i.v.) injected with Saline, DTX/pD-CA-(PCL-ran-PLA) NPs, and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs at the same volume and dose. After 12 h of injection, NIR laser irradiation was performed (808 nm, 1.5 W/cm2 , 10 min) at tumor sites. The temperature changes of tumor sites, as well as infrared thermographic maps, were recorded by an infrared thermal imaging camera (TI100 Infrared Camera FLK-TI100 9HZ, FLUKE).

For in vivo antitumor efficacy studies, Apt-pD-CA-(PCL-ran-PLA) NPs were chosen based on their excellent performance on in vitro cellular targeting, in vitro antitumor efficacy, and in vivo tumor targeting. Different treatments were performed when the tumor volume reached approximately 100 mm3 . The tumor-bearing mice were randomly divided into five groups

(n = 5), and different treatments were performed on Day 0, 4, 8, and 12: (1) Saline, (2) Taxotere R , (3) DTX/Apt-pD-CA-(PCLran-PLA) NPs, (4) Apt-pD-CA-(PCL-ran-PLA) NPs + NIR, and (5) DTX/Apt-pD-CA-(PCL-ran-PLA) NPs + NIR. All the formulations were i.v. injected. Taxotere R and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs were injected at a same DTX dose of 10 mg/kg in 100 µL PBS. Apt-pD-CA-(PCL-ran-PLA) NPs were injected at a same NP dose with DTX/Apt-pD-CA-(PCLran-PLA) NPs. NIR laser irradiation was performed (808 nm, 10 min, 1.5 W/cm2 ) after 12 h injection of NPs. The tumor size and body weights were recorded every 2 days. After 14 days of treatment, the mice were humanely executed. Tumor growth profiles were recorded to evaluate the antitumor efficacy in this study.

Another batch of SCID mice bearing MCF-7 tumor xenograft were further used to access the antitumor efficacy of the mice through recording their survival time after receiving the same treatments mentioned above. The mice with similar physical status (i.e., age, body weight, and 100 mm3 of tumor volume) were randomly divided into five groups (n = 5). The survival results were presented as Kaplane–Meier plots and evaluated using a log-rank test.

# Statistical Methodology

All tests were performed at least three times in all studies unless otherwise stated. The results are expressed as mean ± SD, and the statistical significance of the results was determined by the Student's t-test. The results were considered to be statistically significant if P < 0.05.

# RESULTS AND DISCUSSION

# Preparation of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs

The star-shaped random copolymer CA-(PCL-ran-PLA) was synthesized through a the ring opening copolymerization of ε-caprolactone (CL) and D,L-lactide (LA) with colic acid (CA) as an initiator (**Figure 1A**) (Tao et al., 2014). The fabrication of DTX-loaded Apt-pD-CA-(PCL-ran-PLA) NPs (DTX/Apt-pD-CA-(PCL-ran-PLA) NPs) was illustrated in **Figure 1B**: (i) First, a modified nano-precipitation method, which is a mild and effective way to prepare drug-loaded NPs (Zeng et al., 2013a, 2015b), was applied to prepare DTX/CA-(PCL-ran-PLA) NPs. (ii) Second, the prime-coating of pD layer was achieved through an oxidative polymerization reaction (pH 8.5) to prepare DTX/pD-CA-(PCL-ran-PLA) NPs. (iii) Finally, conjugation of aptamers on the surface of DTX/pD-CA-(PCL-ran-PLA) NPs could be fulfill via a Michael addition reaction in a weak alkaline solution (pH 8.0). The suspensions gradually turned darker in the second step after dopamine hydrochloride was added, indicating the formation of pD layer. A 26-mer SH-terminated DNA aptamer-AS1411, which possesses a thiol group at the 30 - end for effective conjugation to the pD layer, was applied to provide high affinity to interact with breast cancer cells (Cao et al., 2009).

# Characterization of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs

The size and surface properties of polymeric NPs, which play a vital role on their drug release, endocytosis and in vivo pharmacokinetics (Tao et al., 2016; Behzadi et al., 2017; Ding et al., 2017), have been well-characterized in this study. As shown in **Figures 2a–c**, the surface morphology of DTX/CA-(PCL-ran-PLA) NPs, DTX/pD-CA-(PCL-ran-PLA) NPs, and DTX/AptpD-CA-(PCL-ran-PLA) NPs was characterized through TEM images. It could be obviously observed that spherical films have been deposited on the surface of DTX/pD-CA-(PCL-ran-PLA) NPs and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs, which is quite different from bare DTX/CA-(PCL-ran-PLA) NPs and gives direct evidences on the formation of pD layers. The average size distribution in TEM images is about 80 nm for DTX/CA-(PCL-ran-PLA) NPs, and about 95 nm for DTX/pD-CA-(PCL-ran-PLA) NPs and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs (**Figures 2d–f**). As presented in **Table 1**, the average DLS size of DTX/CA-(PCL-ran-PLA) NPs was 103.4 ± 3.3 nm (PDI 0.126), while that of DTX/pD-CA-(PCL-ran-PLA) NPs was 120.3 ± 4.6 nm (PDI 0.115) and that of DTX/Apt-pD-CA-(PCLran-PLA) NPs was 124.6 ± 5.1 nm (PDI 0.123). The increased DLS size of DTX/pD-CA-(PCL-ran-PLA) NPs and DTX/AptpD-CA-(PCL-ran-PLA) NPs could be attributed to the thickness of pD layers formed on the surface of DTX/CA-(PCL-ran-PLA) NPs, which also indicates the successful modification of pD layers through the oxidative polymerization reaction. With the mean hydrodynamic size of NPs ranging from about 100–120 nm in diameter, they are perfectly fit for high accumulation in tumor vasculature driven by the influence of the enhanced permeability and retention (EPR) effect (Zhu et al., 2015; Zhu X. et al., 2016, 2017; Liu et al., 2017). The smaller size obtained from TEM images (compared with DLS testing) may be contributed to the shrink and collapse of NPs when in the dry state (Tao et al., 2013). The stability of NPs was accessed by monitoring particle size in PBS over a span of 2 weeks (**Figure 3A**). The average size of all the NPs hardly changed during the testing period, indicating a great stability of the NPs.

As shown in **Table 1**, the absolute value of zeta potential of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs (−19.2 ± 5.2 mV) increased slightly or kept similarly compared to DTX/pD-CA- (PCL-ran-PLA) NPs (−18.6 ± 3.6 mV) and DTX/CA-(PCLran-PLA) NPs (−17.8 ± 3.9 mV). The negative charge of NPs could benefit the blood compatibility and passive accumulation of the NPs in tumor sites through reducing clearance by the reticuloendothelial system (RES) including liver. Moreover, **Table 1** also showed that the LC and EE of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs (9.73 ± 0.46%, 94.18 ± 2.76%) were almost the same as those of DTX/pD-CA-(PCL-ran-PLA) NPs (9.98 ± 0.39%, 94.31 ± 1.98%) and DTX/CA-(PCL-ran-PLA) NPs (10.02 ± 0.28%, 95.01 ± 2.16%), indicating this strategy is facile and does not affect the effectiveness of drug LC and EE.

# In Vitro Drug Release Kinetics

**Figure 3B** showed the in vitro DTX release from DTX/CA- (PCL-ran-PLA) NPs, DTX/pD-CA-(PCL-ran-PLA) NPs, and

TABLE 1 | Characterization of DTX/CA-(PCL-ran-PLA) NPs, DTX/pD-CA-(PCL-ran-PLA) NPs and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs.

PDI, polydispersity index; ZP, zeta potential; LC, loading content; EE, encapsulation efficiency, n = 3.

DTX/Apt-pD-CA-(PCL-ran-PLA) NPs in the release medium (pH 7.4, PBS containing 0.1% w/v Tween 80) at 37◦C. An initial burst release of DTX could be observed within the first 48 h, as about 40% of the loaded drugs were released in all kinds of NPs. At the end of our release studies (Day 15), about 66.8, 63.2, and 62.8% of DTX were released from DTX/CA-(PCL-ran-PLA) NPs, DTX/pD-CA-(PCL-ran-PLA) NPs, and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs, respectively. Notably, the release rates from DTX/CA- (PCL-ran-PLA) NPs, DTX/pD-CA-(PCL-ran-PLA) NPs, and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs were quite similar, which means the pD modification and AS1411 functionalization did not actually change the drug release properties of the prepared NPs. Therefore, Apt-pD-CA-(PCL-ran-PLA) NPs may be a promising drug delivery platform in nano-biotechnology and nanomedicines.

# In Vitro Photothermal Properties of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs

The photothermal properties of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs was accessed by testing the temperature changes under the irradiation of an 808-nm NIR laser. **Figures 4A,E** showed a rapid temperature increase of ∼20◦C was achieved by DTX/pD-CA-(PCL-ran-PLA) NPs and DTX/Apt-pD-CA- (PCL-ran-PLA) NPs (500 µg/mL) after 10 min of irradiation with the NIR laser (1.0 W/cm2 ). However, DI water and the DTX/CA-(PCL-ran-PLA) NPs showed little temperature change at totally the same conditions, demonstrating the superior photothermal effect of these NPs was majorly attributed to the pD layers. As shown in **Figure 4B**, the photothermal effect of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs had a concentrationdependent relationship. The solutions of DTX/Apt-pD-CA- (PCL-ran-PLA) NPs also exhibited a laser power intensitydependent manner as the temperatures increased with the rise of laser power intensity (**Figure 4C**). Moreover, the temperature changes did not exhibit noticeable difference during five cycles of "laser on"-"laser off " (**Figure 4D**), indicating an excellent photo-stability of DTX/Apt-pD-CA- (PCL-ran-PLA) NPs. Therefore, DTX/Apt-pD-CA-(PCL-ran-PLA) NPs have a good photothermal effect and a superior photostability, which could be applied in cancer photothermal therapy.

#### Endocytosis of Fluorescent NPs and in Vitro Cellular Targeting

Since the endocytosis and sustained retention of NPs acts as a critical and final step on the therapeutic efficacy of NPs (Xu et al., 2012; Behzadi et al., 2017), the endocytosis of Apt-pD-CA-(PCL-ran-PLA) NPs was carefully investigated. We replaced the loaded DTX with a fluorescent molecule, coumarin-6 (C6), in order to study the endocytosis of all the developed NPs in MCF-7 cells. The CLSM images of MCF-7 cells after 2 h of incubation with 250 µg/mL of C6/CA-(PCL-ran-PLA) NPs, C6/pD-CA-(PCL-ran-PLA) NPs, and C6/Apt-pD-CA-(PCL-ran-PLA) NPs in DMEM are provided in **Figure 5A**. As could be observed from this figure, the fluorescence intensity (i.e., representing cellular uptake efficiency) within the MCF-7 cells did not show much difference between C6/CA-(PCLran-PLA) NPs and C6/pD-CA-(PCL-ran-PLA) NPs. Notably, the fluorescence intensity increased significantly within the MCF-7 cells incubated with C6/Apt-pD-CA-(PCL-ran-PLA) NPs, demonstrating the presence of AS1411 aptamers on NP surface may lead to high levels of in vitro cellular targeting efficacy.

In order to quantitatively verify the significant role of AS1411 aptamers during the endocytosis of NPs, MCF-7 cells were incubated with C6/CA-(PCL-ran-PLA) NPs, C6/pD-CA- (PCL-ran-PLA) NPs, and C6/Apt-pD-CA-(PCL-ran-PLA) NPs at the concentration of 100, 250, and 500 µg/mL, respectively. As shown in **Figure 5B**, the cellular uptake efficiency of C6/CA-(PCL-ran-PLA) NPs did not show much difference compared to that of C6/pD-CA-(PCL-ran-PLA) NPs at all concentrations. However, the cellular uptake efficiency of C6/Apt-pD-CA-(PCL-ran-PLA) NPs was 1.37-, 1.53-, and 1.91 fold of that of C6/CA-(PCL-ran-PLA) NPs at the concentration of 100, 250, and 500 µg/ml, respectively. In addition, similar results could also be confirmed by FCM assays (**Figure 5C**). Taken all together, the specific interactions between AS1411 aptamers and MCF-7 cells reinforcing the endocytosis of targeted C6/Apt-pD-CA-(PCL-ran-PLA) NPs compared to

non-targeted C6/CA-(PCL-ran-PLA) NPs and C6/pD-CA-(PCLran-PLA).

# Effects of the Developed NPs on Cell Viability

MTT assays were carried out to evaluate the cytotoxicity of all the developed NPs. We first checked the in vitro toxicity of drug-free CA-(PCL-ran-PLA) NPs, pD-CA-(PCL-ran-PLA) NPs, and Apt-pD-CA-(PCL-ran-PLA) NPs at different concentrations (25, 125, 250, and 500 µg/mL) and after different incubation time (24 and 48 h). As shown in **Figures 6A,B**, none of these drug free NPs showed any functionalization of NPs (pD modification and aptamer conjugation) were biocompatible and non-toxic. Furthermore, the in vitro therapeutic efficacy of DTX/CA-(PCL-ran-PLA) NPs, DTX/pD-CA-(PCL-ran-PLA) NPs, and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs without (i.e., chemotherapy groups) and with (i.e., chemo-photothermal therapy groups) the irradiation of NIR laser (808 nm, 10 min, 1.5 W/cm2 ) was performed in MCF-7 cells. Taxotere R , which is a clinical DTX formulation, was chosen as a reference. Group treated with only NIR laser were also chosen as a reference. MCF-7 cells were treated at 0.25, 2.5, 12.5, and 25 µg/mL of equivalent DTX concentrations for 24 and 48 h. For the chemo-photothermal therapy groups, NIR laser irradiation was performed immediately after adding different NPs before 24 or 48 h of incubation. It could be concluded from **Figure 6C**: (i) the cell viability decreased as the incubation time increased for both Taxotere R and NP groups, indicating a time-dependent and dose-dependent effect; (ii) the DTX/Apt-pD-CA-(PCL-ran-PLA) NPs showed better in vitro therapeutic efficacy than Taxotere R , DTX/CA- (PCL-ran-PLA) NPs, DTX/pD-CA-(PCL-ran-PLA) NPs, and DTX/Apt-pD-CA-(PCL-ran-PLA) NPs. For instance, the cell viability of MCF-7 cells (48 h, at the DTX concentration of 12.5 µg/mL) was 48.2% for Taxotere R , 42.3% for DTX/CA- (PCL-ran-PLA) NPs, 39.4% for DTX/pD-CA-(PCL-ran-PLA) NPs, and 25.9% for DTX/Apt-pD-CA-(PCL-ran-PLA) NPs; (iii) the NIR laser irradiation did not change the viability of MCF-7 cells treated with DTX/CA-(PCL-ran-PLA) NPs. However, the cell viability significantly decreased for groups treated with DTX/pD-CA-(PCL-ran-PLA) NPs and DTX/AptpD-CA-(PCL-ran-PLA) NPs after the irradiation of NIR laser. For example, the cell viability of MCF-7 cells (48 h, at the DTX concentration of 12.5 µg/mL) was 42.8% for DTX/CA-(PCLran-PLA) NPs + NIR, 27.9% for DTX/pD-CA-(PCL-ran-PLA) NPs + NIR, and 16.2% for DTX/Apt-pD-CA-(PCL-ran-PLA)

NPs + NIR. (iv) the DTX/CA-(PCL-ran-PLA) NPs and DTX/pD-CA-(PCL-ran-PLA) NPs showed similar viability at various concentrations, further demonstrating the surface coating of pD layers is biocompatible and non-toxic. In a word, AS1411 aptamers may have an effective targeting efficacy for NPs and the synergistic chemo-photothermal strategy showed the most significant therapeutic efficacy. Therefore, DTX/Apt-pD-CA- (PCL-ran-PLA) NPs could be employed as promising targeted drug delivery systems for effective chemo-photothermal cancer therapy.

# In Vivo Photothermal Imaging of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs

Based on the promising in vitro targeting and therapeutic efficacy, we performed in vivo photothermal imaging of mice after intravenous (i.v.) injection of saline,

DTX/pD-CA-(PCL-ran-PLA) NPs, and DTX/Apt-pD-CA- (PCL-ran-PLA) NPs respectively to further access the in vivo targeting efficacy. After 12 h of i.v. injection, NIR laser irradiation (808 nm, 10 min, 1.5 W/cm2 ) was performed at tumor sites. As shown in **Figure 7**, the temperature increments in the tumor sites of the mice treated with DTX/Apt-pD-CA-(PCL-ran-PLA) NPs and DTX/pD-CA-(PCL-ran-PLA) NPs were 20.8◦C and 11.1◦C respectively, reaching a temperature of 53.8◦C and 44.5◦C after irradiation. DTX/Apt-pD-CA-(PCL-ran-PLA) NPs group exhibited a higher temperature increment than DTX/pD-CA-(PCL-ran-PLA) NPs group, which could be explained by the active tumor targeting effect of AS1411 aptamers and higher accumulation of DTX/Apt-pD-CA-(PCL-ran-PLA) NPs in tumor sites. However, the tumor site temperature of mice

received i.v. injection of saline did not show much increment with a highest temperature of 36.8◦C, which was not enough for tumor ablation.

# In Vivo Antitumor Effects of NPs

The blood circulation time was firstly accessed by in vivo pharmacokinetic studies before in vivo antitumor studies. As shown in Supplementary Figure S1, all the prepared NPs have extended the blood circulation time of the DTX compared to Taxotere R , which may contribute to effective delivery of DTX in vivo. On the basis of the above in vitro cell experiments and in vivo targeting imaging studies, DTX/Apt-pD-CA-(PCLran-PLA) NPs were chosen to further verify the in vivo antitumor efficacy of this targeting chemo-photothermal strategy. As shown in **Figures 8A,B**, both commercial Taxotere R and NP treatment groups could inhibit the tumor growth. In detail, all the NP treatment groups, i.e., DTX/Apt-pD-CA-(PCL-ran-PLA) NPs (target chemotherapy group), Apt-pD-CA-(PCL-ran-PLA) NPs + NIR (target photothermal group), and DTX/Apt-pD-CA- (PCL-ran-PLA) NPs + NIR (target chemo-photothermal therapy group), showed better in vivo antitumor efficacy than Taxotere R . The synergistic chemo-photothermal therapy group showed the highest therapeutic efficacy which significantly eliminated the tumors. These results were consistent with the above cellular experiments and in vivo targeting studies. Moreover, the body weight of the mice in all the NP treatment groups did not show any difference compared to the control group receiving saline, which that of Taxotere R showed slightly weight loss, indicating the potential in vivo biocompatibility of DTX/AptpD-CA-(PCL-ran-PLA) NPs (**Figure 8C**). Another batch of nude mice were used to access the survival data of nude mice receiving different treatments (**Figure 8D**). As could be concluded from this figure, the survival time of "DTX/Apt-pD-CA-(PCL-ran-PLA) NPs + NIR" group was at least extended by 35, 28, 14, and 11 days compared to "Saline" group, "Taxotere R " group, "DTX/Apt-pD-CA-(PCL-ran-PLA) NPs" group, and "Apt-pD-CA-(PCL-ran-PLA) NPs + NIR" group. These results further demonstrated the significant therapeutic efficacy of this target chemo-photothermal therapy strategy based on DTX/Apt-pD-CA-(PCL-ran-PLA) NPs, indicating these NPs are able to be applied as promising targeting drug delivery systems for synergistic chemo-photothermal therapy of breast cancer.

# CONCLUSION

In this study, we for the first time reported the successful synthesis of robust DTX/Apt-pD-CA-(PCL-ran-PLA) NPs with star shaped CA-(PCL-ran-PLA) copolymers, which could be applied as promising targeting drug delivery systems for synergistic chemo-photothermal therapy of breast cancer. By surface modification with facile dopamine polymerization method, AS1411 aptamers were able to be simply conjugated on the surface of NPs for target delivery of drugs. The DTX/AptpD-CA-(PCL-ran-PLA) NPs were well characterized by surface morphology, LC and EE, stability, photothermal properties and drug release profiles. The in vitro and in vivo targeting effect of these NPs were also accessed. The in vitro cytotoxicity assays by MTT showed that DTX/Apt-pD-CA-(PCL-ran-PLA) NPs together with NIR laser irradiation (target chemo-photothermal therapy) could significantly inhibit cell proliferation compared with all other groups. The in vivo antitumor assays, as well as the improved survival time and reduced side effects, further confirmed the significant therapeutic effects of this target chemophotothermal therapy strategy. All the results observed from the in vivo studies were consist with the in vitro assays, suggesting the robust DTX/Apt-pD-CA-(PCL-ran-PLA) NPs are promising in target delivery of drugs and synergistic chemo-photothermal therapy of breast cancer.

### AUTHOR CONTRIBUTIONS

fphar-09-00125 February 20, 2018 Time: 16:54 # 12

NK, MD, Y-DC, and X-BS conceived the idea and designed the study. NK and MD performed all the experiments and analyzed the data. X-NS helped in nanoparticle preparation and in vitro experimental assays. Y-DC and X-BS provided the technical support and corrections of the manuscript. NK wrote the manuscript and revised it according to the comments of Y-DC and X-BS.

# REFERENCES

#### FUNDING

This research was supported by grants from National Natural Science Foundation of China (Grant No. 81672932), Zhejiang Provincial Natural Science Foundation of China for Distinguished Young Scholars (Grant No. LR18H160001), Zhejiang Province Medical Science and Technology Project (Grant No. 2017RC007), Talent Project of Zhejiang Association for Science and Technology (Grant No. 2017YCGC002), and Zhejiang Province Science and Technology Project of TCM (Grant No. 2015ZB033).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2018.00125/full#supplementary-material

and minimally invasive pharmacokinetic analysis. Nanoscale 9, 13465–13476. doi: 10.1039/c7nr02363d

microfilament imaging in living cells. Chem. Commun. 53, 7541–7544. doi: 10.1039/C7CC02555F

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kong, Deng, Sun, Chen and Sui. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Significant Suppression of Non-small-cell Lung Cancer by Hydrophobic Poly(ester amide) Nanoparticles with High Docetaxel Loading

Xing Chen1† , Lili Zhao2† , Yang Kang3† , Zhiyu He1 , Fei Xiong1 , Xiang Ling1 \* and Jun Wu1 \*

#### Edited by:

Jianxun Ding, Changchun Institute of Applied Chemistry (CAS), China

#### Reviewed by:

Guoming Sun, Sunogel Biotechnologies, Inc., United States Yue Pan, Soochow University, China Weiping Wang, The University of Hong Kong, Hong Kong

#### \*Correspondence:

Jun Wu<EMAIL_ADDRESS>Xiang Ling<EMAIL_ADDRESS>
†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology

> Received: 18 January 2018 Accepted: 01 February 2018 Published: 28 February 2018

#### Citation:

Chen X, Zhao L, Kang Y, He Z, Xiong F, Ling X and Wu J (2018) Significant Suppression of Non-small-cell Lung Cancer by Hydrophobic Poly(ester amide) Nanoparticles with High Docetaxel Loading. Front. Pharmacol. 9:118. doi: 10.3389/fphar.2018.00118 1 Key Laboratory of Sensing Technology and Biomedical Instrument of Guangdong Province, School of Engineering, Sun Yat-sen University, Guangzhou, China, 2 Digestive Endoscopy Center, Jiangsu Province Hospital, Nanjing, China, 3 Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China

Non-small-cell lung cancer (NSCLC) accounts for over 85% of clinical lung cancer cases, which is the leading cause of cancer-related death. To develop new therapeutic strategy for NSCLC, a library of L-phenylalanine-based poly(ester amide) (Phe-PEA) polymers was synthesized and assembled with docetaxel (Dtxl) to form Dtxl-loaded Phe-PEA nanoparticles (NPs). The hydrophobic Phe-PEA polymers were able to form NPs by nanoprecipitation method and the characterization results showed that the screened Dtxl-8P4 NPs have small particle size (∼100 nm) and high Dtxl loading (∼20 wt%). In vitro experiments showed that Dtxl-8P4 NPs were rapidly trafficked into cancer cells, then effectively escaped from lysosomal degradation and achieved significant tumor cell inhibition. In vivo results demonstrated that Dtxl-8P4 NPs with prolonged blood circulation could efficiently deliver Dtxl to A549 tumor sites, leading to reduced cell proliferation, block metastasis, and increase apoptosis, then persistent inhibition of tumor growth. Therefore, Phe-PEA NPs are able to load high amount of hydrophobic drugs and could be a promising therapeutic approach for NSCLC and other cancer treatments.

 More than 60% of NSCLC patients are diagnosed to have advanced or metastatic tumors, which are unsuitable for surgical resection with curative intent (Tong, 2006; Pao and Chmielecki, 2010). Thus, alternative therapeutic platforms to control or inhibit tumor development are highly desired. Docetaxel (Dtxl) is of the chemotherapy drug class taxane, structurally similar to paclitaxel, but more effective as the inhibitor of microtubule depolymerization (Bissery, 1995). In the past decades, Taxotere has emerged as one of the most important cytotoxic agents, with proven clinical efficacy against many cancers including NSCLC (Kintzel et al., 2006; Baker et al., 2009). However, the use of

**52**

Dtxl in this formulation with non-ionic surfactant Tween 80 and 13% ethanol leads to several well-known adverse reactions due to either the agents itself (e.g., neutropenia, anemia, nephrotoxicity, neurotoxicity, and musculoskeletal toxicity) or the solvent system (e.g., hypersensitivity and fluid retention) (Persohn et al., 2005). Side effects of commercial Taxotere have considerably overshadowed its clinical application.

Nanoparticles (NPs) have been extensively reported for their prominent superiorities that can be delivered to specific sites by size-dependent passive targeting (Matsumura and Maeda, 1986; Pan et al., 2011a,b, 2012; Bertrand et al., 2014; Deng et al., 2016; Lu et al., 2016; Zhao et al., 2016; Li et al., 2017; Lin et al., 2017; Liu et al., 2017; Shi et al., 2017). Among them, numerous nanoplatforms have been utilized to deliver Dtxl for improved cancer treatment (Tao et al., 2014; Bowerman et al., 2017; Laura et al., 2017). However, the clinical application of most reported platforms is hindered by the low loading capacity of Dtxl (Guo and Huang, 2014). Thus, the carrier material with reasonable hydrophobicity is urgently needed (Chu et al., 2013). Amino acid-based poly(ester amide) polymers with both ester and amide blocks on their backbones have been widely studied over many years (Wu et al., 2012a,b; Wu and Chu, 2013), as they possess not only good biodegradability and biocompatibility but also tunable physicochemical properties, especially hydrophobicity (Wu et al., 2011, 2015; Yu et al., 2014), which may be promising for developing NPs with high Dxtl loading.

Hence, we postulated that the novel design of hydrophobic L-phenylalanine-poly(ester amide) (Phe-PEA) polymer NPs with higher Dtxl loading could bring about more effective antitumor efficiency with better in vivo tolerance. In this paper, Phe-PEA polymers comprised of phenylalanine, diacid, and diol were synthesized and used because of their excellent NP formation and drug loading capability. Dtxl-loaded Phe-PEA polymer NPs were prepared by nanoprecipitation and the physicochemical characteristics were determined. After optimization, Dtxl-8P4 NPs with attractive uptake kinetics and strong cytotoxicity were found to greatly improve circulation retention, then enhance therapeutic effects for A549 tumors with less systemic toxicity (**Figure 1**).

# MATERIALS AND METHODS

#### Materials and Reagents

L-Phenylalanine, 1,4-butanediol, 1,6-hexanediol, adipoyl dichloride, sebacoyl dichloride, toluene-4-sulfonic acid monohydrate, and p-nitrophenol were purchased from Sigma–Aldrich and used without further purification. DMSO, acetone, toluene, triethylamine, ethyl acetate, and methanol were purchased from Aladdin. Dtxl was purchased from LC Laboratories. Taxotere R was purchased from Sanofi Aventis. Dil was purchased from Thermo Fisher Scientific, United States. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy- (polyethylene glycol)-3000] (ammonium salt) (DSPE-PEG 3000) was purchased from Avanti Polar Lipids. F-12K, EMEM, trypsin-EDTA, fetal bovine serum (FBS), penicillin–streptomycin solution, phosphate-buffered saline (PBS), and water were provided by Gibco R .

#### Synthesis of Monomers and Polymers

The synthesis of Phe-PEA polymers was divided into following steps (Katsarava et al., 1999): (i) preparation of di-p-nitrophenyl esters of dicarboxylic acid (monomer I) by condensation reaction; (ii) preparation of toluene-4-sulfonic acid salts of bis(Phe) alkylene diesters (monomer II) via solid–liquid reaction; (iii) preparation of Phe-PEA polymers through solution polycondensation (**Figure 2**). Synthetic details of monomers I and II could be found in previous reports (Wu and Chu, 2012), while Phe-PEA polymers (yield > 80%) were obtained by optimized protocols (Fonseca et al., 2014): monomers I (5 mmol) and II (5 mmol) in dry DMSO (8 ml) were mixed well by vortex and kept at 120◦C under vigorous stirring, then triethylamine (15 mmol) was drop-wisely added to get a uniform yellow solution. The mixture was kept at 80◦C overnight without stirring and resulting polymers were precipitated by adding cold ethyl acetate, washed with methanol, and dried under vacuum.

Here, two kinds of monomers I were prepared: di-pnitrophenyl adipate (N4, x = 4) and di-p-nitrophenyl sebacate (N8, x = 8). Two kinds of monomers II were prepared: toluene-4-sulfonic acid salts of bis(Phe) butane diesters (Phe-4, y = 4) and toluene-4-sulfonic acid salts of bis(Phe) hexane diesters (Phe-6, y = 6). Phe-PEA polymers (x-Phe-y) were prepared by solution polycondensation of monomers I and II at various combinations and summarized in **Table 1**, where x was the numbers of methylene in diacid and y was the numbers of methylene in diol. Chemical structures of above monomers and polymers were confirmed by 1H-NMR (Avance III, Bruker, Switzerland). All the spectra were the same as previously reported (**Supplementary Figure S1**) (Katsarava et al., 1999).

For measuring of molecular weight (MW) of Phe-PEAs, gel permeation chromatography (GPC) method was used and the PEA solutions were prepared at a concentration of 1 mg/ml in a tetrahydrofuran (THF) solution. The MWs of Phe-PEAs were determined from a standard curve generated from polystyrene standards with MWs ranging from 841.7 to 2.93 kDa that were chromatographed under the same conditions as the samples. The standard curve was generated from a third-order polynomial fit of the polystyrene standard MWs (**Table 2**).

#### Preparation and Characterization of Nanoparticles (NPs)

Dtxl-8P4 NPs were prepared by nanoprecipitation method: 6 mg of 8-Phe-4 polymer and a certain amount of Dtxl (10, 20, or 30 wt% of NPs) was dissolved in 0.2 ml of DMSO. Next, the mixture was dropwise added to 10 ml of aqueous solution containing DSPE-PEG 3000 (20 wt% of

TABLE 1 | Phe-PEA polymers and their corresponding NPs.

TABLE 2 | Molecular mass characteristics of Phe-PEA polymers.

NPs) under vigorously stirring. The remaining free molecules and organic solvent were removed by washing with PBS twice using Amicon Ultra-15 centrifugal filters (MWCO 100 KDa, Millipore, United States). Finally, Dtxl-8P4 NPs were dispersed in 1.0 ml of PBS for further use. 8P4 NPs were prepared without Dtxl and used as blank control. Dtxl-4P4, Dtxl-4P6, or Dtxl-8P6 NPs were prepared by the similar procedure using 6 mg of 4-Phe-4, 4-Phe-6, or 8-Phe-6 polymer and fixed feeding of Dtxl (10 wt% of NPs). Dil-8P4 NPs were prepared by mixing pre-determined amounts of 8-Phe-4 polymer, Dtxl, and Dil (3 wt% of NPs) in DMSO, then following above nanoprecipitation procedure. Particle size and zeta potential of NPs were measured by dynamic light scattering (DLS, Zetasizer Nano-ZS90, Malvern, United Kingdom). Morphology of NPs was visualized by transmission electron microscopy (TEM, Tecnai G2 Spirit, FEI, United States).

Docetaxel loading capacity of NPs was determined by Agilent 1260 HPLC with a ZORBAX Extend-C18 column at the temperature of 30◦C and a flow rate of 1.0 ml/min (mobile phase, water:acetonitrile = 50:50). The injection volume was 20 µl for each sample. The UV detection wavelength was 232 nm and Dtxl loading was calculated using following equation:

Loading capacity % = Weight of loaded drugs Weight of polymers + Weight of loaded drugs × 100.

#### In Vitro Release Profiles

Dtxl-8P4 NPs were transferred to dialysis bags (MWCO 3500 Da, Spectrum, United States) and immersed in PBS (pH 5.0 or 7.4). Dtxl release was conducted at 37◦C with constant stirring at 100 rpm. At specific time intervals, 1 ml of the sample solution was collected and replaced with equal volume of fresh PBS.

The amounts of Dtxl were analyzed by UPLC-MS/MS (TSQ Quantum Access Max, Thermo Fisher Scientific, United States) with following MS ionization parameters: positive ESI mode; spray voltage, 3500 V; ion source temperature, 300◦C; collision energy, 0 eV. The analytes were quantified by using Multiple Reaction Monitoring (MRM) to monitor ion transitions m/z of 830.2–303.7. Chromatography was performed via Agilent 1100 HPLC with an Ultimate XB-C18 column at the temperature of 40◦C and a flow rate of 0.2 ml/min (mobile phase, 0.1% formic acid:methanol = 40:60). The gradient elution was 60% methanol at 0–0.30 min, 60–100% methanol at 0.30–0.50 min, 100% methanol at 0.50–2.00 min, 100–60% methanol at 2.00– 2.50 min, and 60% methanol at 2.50–5.00 min.

#### Cell Culture

A549, PC3, and DU145 cells were purchased from American Type Culture Collection (ATCC) and cultured by recommended protocols from the manufacturer. Cells were grown in the corresponding medium, supplemented with 10% FBS and 1% penicillin–streptomycin solution, maintained at 37◦C and 5% CO2.

#### Cellular Uptake

A549 cells were seeded in six-well plates (20,000 cells per well) and incubated with 1 ml of complete medium for 24 h. Dil-8P4 NPs at different concentrations were added. At selected time points, cells were washed with cold PBS twice, harvested by trypsinization, centrifuged, and resuspended in 4% formaldehyde, then analyzed by flow cytometer (FACSCalibur, BD, United States).

#### Cellular Internalization

A549 cells were seeded in 35-mm dishes (20,000 cells per well) and incubated with 1 ml of complete medium for 24 h. Dil-8P4 NPs were added. At selected time points, cells were washed with cold PBS twice and fixed with 4% formaldehyde at 37◦C for 15 min. Subsequently, cells were washed with PBS twice again and stained with LysoTracker green and Hoechst 33342, then observed under an FV3000 confocal laser scanning microscope (CLSM, Olympus, Japan).

#### Cytotoxicity

Cytotoxicity was evaluated with AlamarBlue Cell Viability Assay (Thermo Fisher Scientific, United States) against A549, PC3, and DU145 cells. Cells were seeded in 96-well plates (5000 cells per well) and incubated with 0.1 ml of complete medium for 24 h. Cells were incubated with different concentrations of 8P4 NPs, Taxotere, or Dtxl-8P4 NPs. At selected time points, cells were treated according to manufacturer's protocol using a microplate reader (Synergy4, Bio Tek Instruments, United States).

#### Animals

BALB/c mice (male, 4–5 weeks old), nude mice (female, 4 weeks old), and SD rats (male, 200–220 g) were provided by the Laboratory Animal Center of Sun Yat-sen University. This animal study was carried out in accordance with the recommendations of "the guidelines of the Experimental Laboratory Animal Committee of Sun Yat-sen University and the National Institutes of Health's Guide for the Care and Use of Laboratory Animals."

TABLE 3 | Dtxl-8P4 NPs at different Dtxl feeding.

The animal protocol was approved by the "Experimental Laboratory Animal Committee of Sun Yat-sen University."

The human pulmonary carcinoma xenograft model was established by subcutaneously injecting A549 cell suspension (2,000,000 cells in medium and Matrigel) into the back region of nude mice. As the volume of xenograft tumor reached ∼100 mm3 , mice were used for following experiments.

#### Pharmacokinetics

SD rats (n = 3 per group) were intravenously injected with either (i) PBS, (ii) Taxotere, or (iii) Dtxl-8P4 NPs at a dose of 5 mg Dtxl/kg, respectively. At pre-determined time points, blood was withdrawn from retro-orbital plexus and plasma was collected. Dtxl concentrations were analyzed by UPLC-MS/MS with the same chromatographic condition as described above. Hundred microliters of plasma was mixed with 10 µl of Dtxl, followed by adding 500 µl of methyl tert-butyl ether, vortexed, and centrifuged at 12,000 rpm for 10 min. The supernatant was evaporated and re-constituted with mobile phase. Pharmacokinetic parameters were calculated with Phoenix WinNonlin 6.3 program (Pharsight Corporation, St. Louis, MO, United States).

#### Histology Analysis

BALB/c mice (n = 5 per group) was intravenously injected with either (i) PBS, (ii) 8P4 NPs, (iii) Taxotere, or (iv) Dtxl-8P4 NPs at a dose of 5 mg Dtxl/kg every 7 days. After 39 days, all the mice were sacrificed and major organs (heart, liver, spleen, lung, kidneys) were excised for hematoxylin and eosin (HE) staining. Slides were observed under a fully automated upright microscope (DM6000 B, Leica, Germany).

#### In Vivo Antitumor Efficacy

A549 tumor-bearing nude mice (n = 5 per group) was intravenously injected with either (i) PBS, (ii) 8P4 NPs, (iii) Taxotere at a maximum tolerated dosage (MTD) of 5 mg Dtxl/kg, (iv) Dtxl-8P4 NPs at a dose of 5 mg Dtxl/kg, or (v) Dtxl-8P4 NPs at a dose of 10 mg Dtxl/kg every 7 days. Body weights and tumor volumes were recorded every 2 days. Tumor volume was calculated as follows:

$$\text{Volume} = \text{length} \times \text{width}^2 / 2.$$

#### HE, IHC, and TUNEL

A549 tumor-bearing nude mice (n = 5 per group) was intravenously injected with either (i) PBS, (ii) 8P4 NPs, (iii) Taxotere, or (iv) Dtxl-8P4 NPs at a dose of 5 mg Dtxl/kg every 7 days. After 39 days, tumors were quickly excised for HE and IHC. Slices were incubated with primary antibodies of CD31 and MMP2 (Cell Signaling) and HRP/DAB Detection IHC Kit (Abcam) according to the manufacturers' instructions.

(D) PC3 cells after exposure for 48 h.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) was performed according to the instruction of In Situ Cell Death Detection Kit (Roche).

#### Statistical Analysis

Results were expressed as mean ± SD and repeated at least three times. Two-tailed Student's t-test was applied to analyze the statistical significance of difference between two groups, oneway analysis of variance (ANOVA) was used for multiple groups. Statistical significance was set at ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

#### RESULTS

#### The Hydrophobic Nature of Phe-PEA Polymers Induced Formation of Dtxl-8P4 NPs with Small Particle Size and High Dtxl Loading

L-Phenylalanine-based poly(ester amide) polymers were prepared by solution polycondensation of monomers I and II at various combinations. By introducing diacid or diol segments with different alkyl chains, the hydrophobic nature of Phe-PEA polymers changed accordingly. As the length of alkyl chain increased, the hydrophobicity as well as loading capacity of polymers enhanced, while the formed NPs tended to possess smaller size, which might be caused by the formation of more dehydrated and compacted cores via hydrophobic force. Due to high drug loading, satisfying entrapment efficiency and reliable stability, Dtxl-8P4 NPs were selected for the following experiments (**Table 3**).

Transmission electron microscopy images showed that the loading of a relatively higher amount of Dtxl (∼16 wt%) into 8P4 NPs did not significantly alter the spherical morphology of NPs, but slightly increased their particle size (**Figures 3A–C**).

#### Dtxl-8P4 NPs Exhibited Attractive Uptake Kinetics and Strong Cytotoxicity in Vitro

In vitro release profiles were obtained by representing the percentage of Dtxl released with respect to the amount of drug loaded into NPs. **Figure 3D** demonstrated a sustained-release phase, in which ca. 82.3 and 96.5% of Dtxl were released from Dtxl-8P4 NPs in 144 h at pH 7.4 and 5.0, respectively. This

sustained release could mainly result from the erosion and degradation of the components of NPs. Importantly, no burst effect was observed, further confirming that Dtxl incorporated into 8P4 NPs was likely to remain association with NPs and be taken up into cells as the form of particles rather than free drugs.

Cellular internalization of NPs was performed by labeling A549 cells with a specific fluorescent probe, Dil, which was entrapped into 8P4 NPs at a minor amount to minimize the effect on intracellular trafficking. As displayed in **Figure 4A**, prolonging incubation time or increasing incubation concentration of Dil-8P4 NPs resulted in higher internalization, suggesting timeand concentration-dependent manners. After trans-membrane transport, Dil-8P4 NPs were found to co-localize with endosomes within 1 h, confirming a relatively fast cellular uptake (**Figure 5**). After that, the red fluorescence was mainly observed in cytoplasm, effectively avoiding the fate of lysosomal degradation. As a next step, in vitro cytotoxicity of Dtxl-8P4 NPs was tested against A549, PC3, and DU145 cells by AlamarBlue (**Figures 4B–D**). The viability of cells treated with Dtxl-8P4 NPs did not demonstrate a significant difference compared with Taxotere at low drug levels, but the inhibiting activity of NPs increased at high drug levels. In addition, blank 8P4 NPs without Dtxl had a negligible toxic effect at all test concentrations.

### Dtxl-8P4 NPs Enhanced Therapeutic Effects for A549 Tumors with Less Systemic Toxicity

In order to verify whether Dtxl-8P4 NPs impaired major organs, pathological examination was evaluated in healthy BALB/c mice (**Figure 6**). Taxotere caused severe hepatotoxicity with several structural and metabolic changes, i.e., vacuolar degeneration

TABLE 4 | Pharmacokinetic parameters of Dtxl after intravenous injection of Dtxl-8P4 NPs or Taxotere.

and inflammatory cell infiltration; splenic nodule atrophy; pulmonary hemorrhage; and tubular dilation with flattening of renal epithelium cells. However, no any noticeable histological alternations were captured in Dtxl-8P4 NPs as well as PBS and 8P4 NPs groups, confirming good biocompatibility.

The plasma Dtxl concentrations vs. time profiles were shown in **Figure 7A**. Pharmacokinetics presented the remarkably enhanced retention of Dtxl-8P4 NPs in blood circulation, whereas Taxotere exhibited the rapid elimination from circulation system. Non-compartmental and twocompartmental analysis showed significant changes in pharmacokinetic parameters of Dtxl (**Table 4**). Area under the curve (AUC0→inf), area under the first moment curve (AUMC0→inf), and mean residence time (MRT0−inf) of NPs

FIGURE 7 | (A) Pharmacokinetics after intravenous injection of Dtxl-8P4 NPs and Taxotere at a dose of 5 mg Dtxl/kg (n = 3 per group). (B) Tumor growth and (C) body weight curves of A549 tumor-bearing nude mice (n = 5 per group) treated with PBS, 8P4 NPs, Dtxl-8P4 NPs, and Taxotere.

were 4.7-, 7.6-, and 1.6-fold higher than Taxotere, while clearance (CL) and volume of distribution (Vss) were reduced by 78.6 and 68.1%, respectively. t1/2 of distribution and elimination phase was all dramatically extended compared with Taxotere.

To prove the potential of Dtxl-8P4 NPs for tumor growth suppression, a schedule of multiple dosing was applied since day 9 after A549 tumor implantation (**Figures 7B,C**). PBS and 8P4 NPs groups exhibited rapid tumor growth, whereas MTD for weekly dosing of Taxotere significantly delayed tumor growth. In comparison, a better tumor inhibition with sustaining weight gain was observed in mice receiving equal dosing of Dtxl-8P4 NPs. What's more, the most aggressive treatment with double dosing of chemotherapy suppressed tumor growth for longer but barely induced weight loss, probably due to biocompatible Dtxl-8P4 NPs prevented the random drug release in the body and enhanced therapeutic efficacy of Dtxl.

#### Dtxl-8P4 NPs Effectively Suppressed Proliferation, Metastasis, and Apoptosis of Tumors

The pathology of tumor tissues revealed coincident results (**Figure 8**). For the vigorous growth of tumors in PBS and 8P4 NPs groups, nuclei and cytoplasm presented a blue– pink interlaced image on the whole section. Once tumors underwent apoptosis, nuclei disappeared and cytoplasm became

an amorphous mass of necrotic material. The destructed tumor area of Dtxl-8P4 NPs accounted for the highest percentage among all the groups, further revealing the enhanced chemotherapeutic efficiency of NPs. Besides, the histological analysis of proliferation, metastasis, and apoptosis for tumors treated with rounds of chemotherapy was carried out through IHC and TUNEL. Dtxl-8P4 NPs greatly suppressed the expression of CD31 and MMP2, compared with other groups, verifying that tumor proliferation and metastasis were effectively restricted (Giatromanolaki et al., 1997; Bjorklund and Koivunen, 2005; Kim et al., 2009; Jacob and Prekeris, 2015). The administration of PBS or 8P4 NPs caused negligible TUNEL-positive staining, while Dtxl-8P4 NPs resulted in the most remarkable apoptosis of tumors, emphasizing the great efficacy of NPs.

#### DISCUSSION

In summary, amino acid-based Phe-PEA polymers were synthesized and formulated with Dtxl to construct Dtxl-loaded Phe-PEA polymer NPs. The hydrophobic nature of polymers contributed to the installation of high hydrophobic payloads. Dtxl-8P4 NPs showed the small particle size ∼100 nm with high loading capacity ∼20 wt%, a low burst effect, and a sustained drug release in vitro. Cytotoxicity of Dtxl-8P4 NPs against tumor cells was superior to Taxotere would be attributed to the rapid cellular uptake and effective lysosomal escape. The better antitumor efficacy and less systemic toxicity of Dtxl-8P4 NPs might be attributed to extended blood circulation and high Dtxl loading. Thus, Dtxl-8P4 NPs could be promising as a novel formulation of Dtxl in cancer chemotherapy to fight against NSCLC.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

XC and LZ conceived and directed the study. YK, ZH, and FX performed syntheses and spectroscopic studies. XC and YK co-wrote the paper. XL and JW oversaw the project and contributed to the execution of the experiments and interpretation of the results. All authors contributed to the characterizations and discussion and reviewed and approved the final paper.

#### FUNDING

This work was supported by the Thousand Talents Plan for Young Professionals, the National Natural Science Foundation of China (21704104), West Light Foundation of the Chinese Academy of Sciences (Y7C1021100), the Science and Technology Planning Project of Guangdong Province (2016A010103015), and the Science and Technology Program of Guangzhou<PHONE_NUMBER>94).

#### ACKNOWLEDGMENTS

We appreciate the valuable and insightful thoughts from all the members of the Wu Laboratory.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2018.00118/full#supplementary-material

FIGURE S1 | 1H-NMR of Phe-6 (solvent: DMSO-d6).

chronic angiogenesis. Am. J. Pathol. 174, 1972–1980. doi: 10.2353/ajpath.2009. 080819

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Chen, Zhao, Kang, He, Xiong, Ling and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Poly(Ethylene Glycol)–Polylactide Micelles for Cancer Therapy

Jixue Wang1,2, Shengxian Li 1,2, Yuping Han3 \*, Jingjing Guan1 \*, Shirley Chung4 , Chunxi Wang1 and Di Li 2 \*

*1 Department of Urology, The First Hospital of Jilin University, Changchun, China, 2 Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China, 3 Department of Urology, China-Japan Union Hospital of Jilin University, Changchun, China, 4 Department of Biology, University of Waterloo, Waterloo, ON, Canada*

#### Edited by:

*Wei Tao, Harvard Medical School, United States*

#### Reviewed by:

*Yingjie Yu, Tufts University, United States Huihui Kuang, Independent Researcher, United States Mahavir Bhupal Chougule, University of Mississippi, United States*

#### \*Correspondence:

*Yuping Han<EMAIL_ADDRESS>Jingjing Guan<EMAIL_ADDRESS>Di Li<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology*

> Received: *12 January 2018* Accepted: *22 February 2018* Published: *08 March 2018*

#### Citation:

*Wang J, Li S, Han Y, Guan J, Chung S, Wang C and Li D (2018) Poly(Ethylene Glycol)–Polylactide Micelles for Cancer Therapy. Front. Pharmacol. 9:202. doi: 10.3389/fphar.2018.00202* For the treatment of malignancy, many therapeutic agents, including small molecules, photosensitizers, immunomodulators, proteins and genes, and so forth, have been loaded into nanocarriers for controllable cancer therapy. Among these nanocarriers, polymeric micelles have been considered as one of the most promising nanocarriers, some of which have already been applied in different stages of clinical trials. The successful advantages of polymeric micelles from bench to bedside are due to their special core/shell structures, which can carry specific drugs in certain disease conditions. Particularly, poly(ethylene glycol)–polylactide (PEG–PLA) micelles have been considered as one of the most promising platforms for drug delivery. The PEG shell effectively prevents the adsorption of proteins and phagocytes, thereby evidently extending the blood circulation period. Meanwhile, the hydrophobic PLA core can effectively encapsulate many therapeutic agents. This review summarizes recent advances in PEG–PLA micelles for the treatment of malignancy. In addition, future perspectives for the development of PEG–PLA micelles as drug delivery systems are also presented.

Keywords: polylactide, poly(ethylene glycol), micelle, nanocarrier, controlled drug release, antitumor treatment

# INTRODUCTION

Cancer is one of the major health problems that threaten human life. According to the worldwide statistic, there were 14.1 million new tumor incidences and 8.2 million cancer-related deaths in 2012 (Scsukova et al., 2015). Cancer cells proliferate uncontrollably and rapidly, thus they are characterized by the development of abnormalities and the combination of mutagenic stages. Moreover, they can realize self-sufficiency in growth signals, resistance to growth inhibition and evasion of apoptotic cues (Luo et al., 2009). Furthermore, tumors can induce angiogenesis, evasion from immune surveillance, and metastasis to distant sites through interactions with surrounding stromal cells (Mohme et al., 2017). All of these reasons have led to the refractoriness of cancer. To date, chemotherapy, radiation, surgery, and hormonal therapies are still the major treatment methods for cancer in clinics. Whereas in the research industry, many other treatments, such as photodynamic therapy, photothermal therapy, gene therapy, immunotherapy, and so forth, are being studied. Despite the relatively satisfactory results these therapeutic agents exhibit, they also possess many disadvantages, including poor pharmacokinetics, unspecific bio-distribution, and low targeting ability. The poor solubility and hydrophobicity are considered the major hurdles when therapeutic agents are applied in cancer therapy. Therefore, it is urgent and necessary to overcome these shortcomings to enhance the anti-tumor efficiency.

With the development of nanotechnology, nanomaterials have been widely used in biological application, such as biosensor, tissue engineering as well as drug delivery (Yu et al., 2016a,b,c; Zhang et al., 2017). Nanocarriers have attracted more and more attention in cancertherapy owing to their unique properties, such as nanoscaled size, high surface-to-volume ratio, and favorable physico-chemical characteristics. Various nanocarriers, including liposomes, micelles, and nanocapsules, have been studied in anticancer trials (He et al., 2016; Hofmann et al., 2017; Niu et al., 2017; Tao et al., 2017). They have the capacity to modulate both pharmacokinetic and pharmacodynamic properties, thereby improving their therapeutic index. Among these nanocarriers, polymeric micelles have gained considerably more attention as a multifunctional drug delivery system for poorly water-soluble agents.

Polymeric micelles are the nano-scaled sized particles (5–200 nm) which are self-assembled by amphiphilic polymers. They consist of two parts: the hydrophobic part on the inside (core) and hydrophilic part on the outside (shell). Therefore, the hydrophobic core can serve as a solubilization depot for agents with poor aqueous solubility. The hydrophilic shell provides advantages including longer blood circulation time and increased stability in the blood. In addition, polymeric micelles can be functionalized with targeting ligands to enhance tumor accumulation. As a result, the role of polymeric micelles in delivery of hydrophobic therapeutic agents for anticancer therapy is promising and opportunistic.

Polyethylene glycol (PEG)-polylactide (PLA) is one of the most prominent amphipathic polymers, therefore it is very suitable for constructing micelles. PLA is a form of biodegradable and biocompatible polyester derived from renewable resources and approved by the Food and Drug Administration (FDA) for clinical use. The hydrophobicity of PLA makes it suitable for the hydrophobic portion of micelles. PLA has three types of stereoisomers: poly(L-(-)-S-lactide) (PLLA), poly(D-(+)-Rlactide) (PDLA), and racemic PDLLA. Interestingly, PLLA and PDLA can form stereocomplexes through physical association of PLLA and PDLA chains (Ikada et al., 1987). PLA can interact with different hydrophilic agents, such as PEG (Danafar et al., 2017), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) (Long et al., 2016), poly(ethylene oxide) (PEO) (Fang et al., 2015), and poly(N-isopropylacrylamide) (PNIPAAm) (Wei et al., 2009), to form amphiphilic block copolymers and further selfassemble into micelles. Among them, PEG is the most popular hydrophilic agent due to its various advantages, including linearity, lack of charge, immunogenicity, low polydispersity, and easy activation for conjugation. PEG–PLA micelles have been widely used as drug delivery systems for cancer therapy owing to the excellent physicochemical and biological properties, namely nontoxicity, non-protein adsorption, and weakened uptake by the reticulo-endothelial system (RES) after intravenous injection (Wang et al., 2015, 2017). Notably, Genexol R - PM, a paclitaxel formulation based on PEG–PLA copolymer micelles, was approved in Korea in 2007 for the treatment of breast, lung, and ovarian cancers (Luo et al., 2012). Moreover, it is currently under clinical development in the USA (Lee et al., 2008).

In this review paper, an overview of PEG–PLA-based micelles utilized for the effective delivery of therapeutic agents possessing varying mechanisms for cancer treatment is discussed, as shown in **Scheme 1**. Additionally, the applications of different treatment modalities are described in detail. Features of nanocarriers in references are shown in **Table 1**. In particular, the development of stimuli-responsive, targeted-modified, and multifunctional PEG–PLA micelles are also highlighted.

# CHEMOTHERAPEUTICS

In clinical settings, surgery and radiotherapy are the most commonly used and effective therapeutic means for local and non-metastatic tumors. However, they are inefficient for metastatic tumors. Currently, application of anti-cancer drugs, such as chemotherapeutic drugs, hormone drugs, and biological drugs, has become the main method of treatment. These anticancer drugs are able to reach all parts of the body via the bloodstream and primarily inhibit the rapid replication of tumor cells. Unfortunately, they also inhibit the rapid growth of healthy cells which are crucial to maintaining normal function of the organism, such as hair follicles, bone marrow, and gastrointestinal tract cells (Chabner and Roberts, 2005). As a result, serious side effects are apparent in chemotherapeutic drug treatments. Due to the severe toxicity and concomitant multidrug resistance of conventional chemotherapeutic drugs, it is urgent to find new effective drug carriers to solve these problems. PEG–PLA micelles provide significant advantages over standard treatments. Drug-loaded micelles exhibit a huge potential for tumor drug delivery to overcome the limitations of chemotherapeutic agents.

As generally known, many chemotherapeutic drugs are not highly water-soluble. Hydrophobic agents are associated with several problems in therapeutic applications, such as poor absorption, bioavailability, and drug aggregation-related complications. Fortunately, polymeric micelles constructed from amphiphilic copolymers can promisingly increase the water solubility of such hydrophobic chemotherapeutic drugs by 10–5000 folds (Savic et al., 2006 ´ ). Puntawee et al. improved the aqueous solubility and bioavailability of semisynthetic andrographolide analog (19-triphenylmethyl ether andrographolide, AG 050) by utilizing the PEG-b-PLA micelles. As a result, PEG-b-PLA micelle was able to significantly increase the encapsulation efficiency of hydrochloride salt of AG 050 (AG 050-P) in aqueous solution (280-fold) (Puntawee et al., 2016).

It is worth noting that conventional chemotherapeutic drugs are quite small in size. As a result, they are rapidly cleared from the bloodstream, thus leading to decreased concentration within the tumor (Allen and Cullis, 2004). When chemotherapeutic drugs are loaded into PEG–PLA micelles, their circulation time in the bloodstream will be prolonged, therefore allowing adequate amounts of drugs to reach the target site (Jin et al., 2017). Furthermore, compared to other drug carriers, micelles have the advantage of possessing a very small size (10– 100 nm). This is critical for passive accumulation through the leaky vasculature and into the tumor site via the enhanced

permeability and retention (EPR) effect to solid tumors, particularly to poorly vascularized tumors (Davis and Shin, 2008; Feng et al., 2017). Two main factors are responsible for the EPR effect: (1) the angiogenic vasculature of tumor sites have higher permeability than normal vasculature because of their discontinuous endothelia and (2) the lymphatic drainage is not fully developed in tumors. These conditions result in the exudation of colloidal particles through the "leaky" endothelial layer of tumor vascular and subsequently accumulating in tumor tissues. Majority of vascular pore cutoff size of tumor sites is between 380 and 780 nm (Hobbs et al., 1998). Shi et al. synthesized a docetaxel (DTX)-conjugated mPEG–PLA micelle via an ester linkage (DTX-PM). The average size of DTX-PM was 58.2 ± 2.3 nm, which was suitable for passive targeting via an EPR effect for anti-cancer drug delivery. More importantly, the DTX-PM significantly induced apoptosis of HSC-3 cancer cells and effectively suppressed the tumor progression in the HSC-3 xenograft model in vivo (Shi et al., 2016) (**Figure 1**). Particle size is a critical factor in the efficacy of drug-loadedmicelles. It is of fundamental importance to understand the association between the size of drug-loaded nanocarriers and their fate in biological systems in vivo for rational design of drug delivery systems. Yu et al. prepared five different sized (30–230 nm) DTX-loaded methoxy poly(ethylene glycol) poly(lactide) micelles. They studied the permeation effect of micelles within both multicellular tumor spheroids and tumor xenografts in mice. The results indicated that small micelles could enhance the tissue penetration however, the blood half-life was short. Micelles of 100–200 nm showed prolonged circulation time compared with that of smaller or larger micelles, but the

#### TABLE 1 | Features of nanocarriers in references.

penetration was limited. Large micelles (>200 nm) had both short half-life and weak penetration ability, leading to limited tumor accumulation (Yu and Qiu, 2016). Through the EPR effect, drug-loaded PEG–PLA micelles can selectively kill tumor cells and reduce the damage to normal tissues.

Active targeting is another effective method to reduce the damage of chemotherapeutic drugs to normal tissues (Allen, 2002; Szakács et al., 2006). Ligands are modified on the surface of nanocarriers to bind to particular receptors overexpressed by tumor cells or tumor vasculature. Many kinds of targeted ligands have been researched, such as small organic molecules, sugar moieties, peptides, and monoclonal antibodies (Allen, 2002; Brannon-Peppas and Blanchette, 2012). Folate is an example of a small organic molecule that is a cancer targeting ligand because the folate receptor is frequently overexpressed on tumors (Cho et al., 2016). Xiong et al. designed folate-conjugated interfacially crosslinked biodegradable micelles consisting of poly(ethylene glycol)-block-poly(acryloyl carbonate)-blockpoly(D,L-lactide) (PEG–PAC–PLA) and folate-PEG–PLA (FA-PEG–PLA) block copolymers for receptor-mediated delivery of paclitaxel (PTX). Remarkably, folate-decorated PTX-loaded crosslinked micelles displayed significantly higher toxicity to KB cells than free PTX. This is most likely due to their much more efficient cellular uptake through FA receptor-mediated endocytosis. Flow cytometry studies also showed that folate-decorated FITC-labeled crosslinked micelles were much more efficiently taken up by KB cells than that of controls without folate ligands. All these results indicated that ligand-conjugated interfacially crosslinked PEG–PLA micelles have great potential in targeted cancer therapy (Xiong et al., 2011). Peptides are another form of actively used ligands in anticancer drug delivery. Li et al. developed DTX-loaded target micelles (c(RGDfK)-PEG–PLA/PEG–PLA/DTX) that were able to targeted delivery DTX to the tumor cells. Cellular uptake and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) studies revealed that the target micelles were more efficiently taken up by HeLa cells in the presence of c(RGDfK) and significantly improved the cytotoxicity compared to that of non-target micelles (Li et al., 2016a). In another study, researchers constructed somatostatin analog octreotide (OCT)-modified, DTX loaded PEG-b-PLA micelles that bind to somatostatin receptors (SSTRs) overexpressed on tumor cells. The results showed that OCT-PM-DTX enhanced intracellular delivery efficiency in cells and exhibited

higher retardation of tumor growth in vivo (Zhang et al., 2011).

In another study, researchers synthesized a folate modified pH sensitive targeted polymeric micelle to reduce the systemic toxicity of doxorubicin (DOX) and to increase the antitumor efficacy in a multi-drug resistant tumor model (Li et al., 2015). A pH sensitive targeted strategy is derived from the pH of the tumor microenvironment which is slightly lower (6.5–7.2) compared to physiological pH (7.4) (Tannock and Rotin, 1989; Gerweck et al., 2006). The triggering mechanisms of most pH sensitive micelles relate to the endosome and release of the loaded drugs into the cytoplasm. This is because the pH significantly drops to 5.0–6.5 in the endosome and to 4.5 in primary and secondary lysosomes (Hubbell, 2003; Schmaljohann, 2006). Therefore, based on the pH of endosomes/lysosomes, pH sensitive PEG– PLA-based micelles loaded with chemotherapeutic drugs have been widely studied to improve the efficiency of cancer therapy. The major strategies used for inducing pH-sensitive behaviors are changing the charges in the micellar system and the dissociating pH-dependent drug binding linkers. Liang et al. constructed size shifting micelle nanoclusters (MNC) based on a cross-linked framework interspersed with PEG–PLA micelles. After being internalized into tumor cells, the framework of MNC became swollen and disintegrated within the acidic environment of lysosomes via the proton sponge effect of PEI resulting in the release of nanosized micelles for further penetration (Liang et al., 2016). Wu et al. fabricated tumor-targeted and pH-responsive polymeric micelles by mixing AP peptide (CRKRLDRN) conjugated PEG-poly(D,L-lactic acid) block copolymer (AP-PEG–PLA) into the pH-responsive micelles of methyl ether poly(ethylene glycol) (MPEG)-poly(-amino ester) (PAE) block copolymer (MPEG-PAE). This micelle showed a sharp pHdependent micellization/demicellization transition at the acidic environment of the tumor. When loaded with DOX, the DOX-loaded micelles exhibited excellent anticancer therapeutic efficacy (Wu et al., 2010).

Redox-responsive nanocarriers are another captivating direction of research for effective intracellular anticancer drug release (Meng et al., 2009; Cheng et al., 2011). Unlike pH-sensitive strategies which usually release drugs in the endo/lysosomal compartments, redox-responsive strategies aim to disassemble and release drugs in the cytosol and cell nucleus where many chemotherapeutic drugs, such as DOX and PTX, elicit their therapeutic effects (Zhang et al., 2012; Chuan et al., 2014). Since the amounts of glutathione (GSH) tripeptide in the cytosol and cell nucleus (∼2–10 mM) are confoundedly higher than that of extracellular fluids and circulation (∼2–20µM), the intracellular redox-response is exceedingly fast and efficient. Yang et al. constructed a type of redox-responsive micelle which could self-assemble from dynamic covalent PEG–PLA block copolymers that contained a double disulfide linkage in the backbone. The in vitro drug release analyses indicated that a reductive environment could result in triggered drug release profiles. A cytotoxicity assay of DOX-loaded micelles indicated higher cellular proliferation inhibition against HeLa cells pretreated with 10 mM GSH monoester (GSH-OEt) for 2 h than that of nonpretreated ones (Yang et al., 2015). In order to further improve the anticancer efficiency of chemotherapeutic drug-loaded PEG–PLA micelles, dual or multi-stimuli responsive micelles that respond to a combination of two or more signals mentioned above have also been developed.

### PHOTOTHERMAL THERAPY COMPONENTS

Photothermal therapy (PTT) has become an effective alternative in cancer therapy because of the advantages over other treatment methods, such as minimal invasiveness, low toxicity, and high specificity to tumor sites (Geng et al., 2015; Mebrouk et al., 2015). In clinical practice, to maximize the light penetration depth and minimize the influence of biological chromophores, photothermal agents should have strong optical absorbance in the near-infrared (NIR) window (700–900 nm) (Jaque et al., 2014). In recent years, a variety of nanomaterials, such as gold nanostructures (nanoshells, nanorods, nanostars and nanocages) (Chen et al., 2010; Zhang et al., 2012; Ma et al., 2013; Wang et al., 2013), carbon nanomaterials (carbon nanotube and graphene) (Liu et al., 2011; Yang et al., 2012a), and various other inorganic (Tian et al., 2011) and organic nanoparticles (Yang et al., 2012b) have been used for PTT. Although most inorganic nanomaterials have shown effective therapeutic effect for cancers, majority of them are non-biodegradable and will retain in the body for a long time (Sharifi et al., 2012; Zhang et al., 2013). Compared to inorganic nanomaterials, polymeric NIR-absorbing nanomaterials agents have shown great superiority for PTT. Among PTT agents, small molecular organic dyes, such as (ICG) (Fang et al., 2017) and prussian blue (PB) (Hoffman et al., 2014), have been used not only as fluorescent probes in optical imaging, but also as PTT agents. Sun et al. fabricated a new photothermal nano-agent by coprecipitation of 2,5-Bis(2,5-bis(2-thienyl)- N-dodecyl pyrrole) thieno[3,4-b][1,2,5] thiadiazole (TPT-TT) and a biodegradable amphiphilic block copolymer, methoxy poly(ethylene glycol)2K-block-poly(D,L-lactide)2K (mPEG2K– PDLLA2K). As a result, TPT-NPs showed high photothermal conversion efficiency, excellent photostability, and heating reproducibility. The photostability of TPT-TT NPs was much better than that of ICG. Besides, TPT-TT NPs exhibited significant photothermal therapeutic effects toward human cervical carcinoma (HeLa) and human liver hepatocellular carcinoma HepG2 cells (Sun et al., 2015) (**Figure 2**).

# PHOTODYNAMIC THERAPY COMPONENTS

Photodynamic therapy (PDT) is a light triggered method for cancer treatment. Due to the minimal aggressiveness and harmlessness to healthy tissue, PDT can avoid the disadvantages of conventional chemotherapeutic agents, such as serious side

FIGURE 2 | (A) Schematic illustration of the preparation and their cellular action process of TPT-TT NPs. (B) TEM image (Scale bar 500 nm) and size distribution determined by DLS of TPT-TT NPs. (C) Photothermal conversion behavior of TPT-TT NPs at various concentrations. (D) Relative cell viabilities of HepG2 cells incubated with different concentrations of TPT-TT NPs. Reproduced with permission from Sun et al. (2015).

effects or multidrug resistances. PDT can kill tumor cells mainly through the generation of singlet oxygen (1O2) or free radicals. The reactive oxygen species (ROS) can cause significant cellular damage, destruction of tumor blood vessels, and stimulation of antineoplastic immunity (Juarranz et al., 2008). PDT depends on three factors to perform its anticancer effect: (i) a photosensitizer (PS) agent; (ii) irradiation of the affected region using light of an appropriate wavelength; and (iii) presence of oxygen. During irradiation, highly reactive species, such as ROS, which are capable of causing direct damage to biomolecules and triggering the death of tumor cells, are generated in situ (Paszko et al., 2011). Recently, the combination of photodynamic therapeutic agents and nanosystems has proven to be promising. Nanosystems, such as liposomes, micelles, and polymeric nanoparticles, have been widely studied for PDT application, as well as for combination of PDT agents with chemotherapeutic agents or with other forms of therapies. For example, Ogawara et al. encapsulated the photoprotoporphyrin IX dimethyl ester (PppIX-DME) into PEG–PLA nanoparticles (PN-Por). The PN-Por showed significant phototoxicity in vitro and effective antitumor effect in C26 tumor-bearing mice in vivo. All results exhibited the potency of PN-Por for PDT-based cancer treatments (Ogawara et al., 2016). In another study, in order to increase the solubility and delivery efficiency of PpIX, Ding et al. prepared PpIX loaded polymeric micelles using non-covalent encapsulation and covalent conjugation methods. Micelles with lower PpIX loading density (e.g., 0.2%) showed brighter fluorescence and higher 1O2 yield than higher PpIX loading density (e.g., 4%) in solution. However, 4% PpIXconjugated micelles demonstrated better antitumor efficiency in vivo (Ding et al., 2011a). Moreover, they further studied the effect of carrier microenvironment on photophysical properties of 5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin (mTHPP) and its biological efficacy in tumor inhibition. The electrondeficient PEG–PLA micelle was used as a control. Results displayed that the photophysical and photodynamic properties of mTHPP were highly related to the micelle core environment (Ding et al., 2011b).

PDT can also be combined with active targeting strategies to enhance the antitumor efficiency. Cyclic RGD (cRGD) is a type of peptide that can target the αvβ3 integrin-rich tumor cells. Tian et al. encapsulated NEt2Br2BDP (a trifunctional photosensitizer) into a cRGD peptide-poly(ethylene glycol)-block-poly(lactic acid) (cRGD-PEG–PLA) and methoxyl poly(ethylene glycol)- blockpoly(lactic acid) (mPEG–PLA) nanomicelle. Under the acidic tumor environment (pH 4.5–5.0), the nanoprobe could be activated to produce fluorescence for tumor detection and 1O2 for effective tumor therapy (Tian et al., 2015) (**Figure 3**).

Although there are few related studies, the PEG–PLA-micellebased PDT represents a new kind of therapeutic method, which may be used in clinics in the future.

#### IMMUNE PREPARATION

Immunotherapy is a method of treatment that utilizes the patients' own immune system to treat their illness. Recent strategies for cancer immunotherapy mainly focus on tumorassociated antigens (TAAs), known as a tumor vaccine, and the induction of antigen-specific T cell-mediated immune responses (Cheever and Higano, 2011; Tefit and Serra, 2011; Ledford, 2014). With the thriving progress of genomics and proteomics, various potential target antigens, such as recombinant proteins, synthetic peptides, and DNA, have been studied (De Gregorio and Rappuoli, 2014). However, when administered alone in vivo, they often suffer from short half-life and ineffectively activate the immune system. Hence, adjuvants are required to elicit effective immune responses against tumor cells (Brichard and Lejeune, 2007). Based on the mechanisms of action, vaccine adjuvants can be divided into two categories: immunomodulatory adjuvants and delivery systems (Huang et al., 2011). Coumes et al. synthesized a peptide/polymer conjugate copolymer by conjugation of the amine end-group of LD-indolicidin to the Nhydroxysuccinimide-activated carboxyl end-group of PEG. When the TAA vaccine candidate was formulated with LD-indolicidin-PEG–PLA-stabilized squalene-in-water emulsion, they could effectively elicit a T helper (Th)1-dominant antigen-specific immune response and exhibit satisfactory antitumor activity (Coumes et al., 2015) (**Figure 4**). To induce T cell-mediated immune responses, Li et al. used PEG–PLA based nanoparticles to deliver cytotoxic T lymphocyte-associated molecule-4 (CTLA−4)-siRNA (NPsiCTLA-4). Both the in vitro and in vivo studies showed that this nanoparticle delivery system could effectively deliver CTLA-4-siRNA into both CD4+ and CD8+ T cells, and could significantly increase the percentage of anti-tumor CD8+ T cells, therefore enhancing the antitumor immune responses (Li et al., 2016b).

#### PROTEINS

Protein therapeutics, especially applying cytokines and antibodies, have attracted increasing attention in recent clinical cancer treatments. Compared to chemotherapeutic drugs, proteins have many unique advantages including high specificity, incorporation of diversified functions, and minor side effects on normal tissue (Leader et al., 2008). The mechanism of protein therapy for cancer treatment typically depends on the direct induction of apoptosis in tumor cells and indirect tumor inhibition by activating an immune response or targeting the tumor vasculature and stroma. In order to overcome the shortcomings of proteins in cancer treatment, such as rapid in vivo degradation, poor pharmacokinetics, and instability, combination of proteins with nano delivery systems has been intensively researched.

Cytokines are a class of secreted or membrane-bound proteins. They play an important role in regulating the growth, differentiation, and activation of immune cells (Dranoff, 2004). Many kinds of cytokines, such as tumor necrosis factors (TNFs), interleukins (ILs), and interferons (INFs), have been widely used in clinical cancer treatment. These cytokines can be targeted through the use of antibodies. Antibodies are one of the most efficient and promising approaches for the treatment of hematological malignant neoplasms and solid

tumors (Scott et al., 2012). A series of monoclonal antibodies (mAbs) have been approved by the FDA or are being assessed in clinical trials for cancer therapy (Scott et al., 2012). Antibodies can also act as target groups to enhance the uptake of drugs in tumor cells and can be combined with a nano drug delivery system. In order to achieve targeted delivery of siPlk1 to Her2+ breast cancer, Dou et al. designed an anti-Her2 single-chain variable fragment antibody (ScFv Her2) decorated PEG–PLA-based nanoparticles encapsulated with siPlk1 (ScFv Her2 -NP si Plk1). This ScFv Her2 -NP siRNA could specifically bind to the Her2 antigen overexpressed on the surface of Her2+ breast cancer cells. Therefore, the antitumor efficiency was evidently improved (Dou et al., 2014). Yue et al. prepared multifunctional hybrid micelles based on amphiphilic mal-PEG-b-PLA and mPEG-b-P(LA-co-DHC/RhB) block copolymers. A specific anti-transferrin receptor antibody, OX26, was then linked onto the surface of the micelles. The results showed that OX26 conjugation visibly increased the uptake efficiency of micelles by target cell lines (C6) and effectively passed through the blood-brain barrier (Yue et al., 2012).

# GENE

Nucleic acid (such as plasmid DNA, antisense oligonucleotides, and siRNA)-based gene therapeutics have received an increased amount of attention in the last few decades because of their unique advantages for attacking critical cancer hallmarks (Das and Verma, 2016; Jiang et al., 2017). Even though the use of viral vectors for gene therapy offers highly efficient gene

transfer and has shown satisfactory results in clinical trials (Alton, 2007), they possess various unwanted effects, such as immune stimulation and the potential for mutagenesis. Therefore, it is clear there is still need for further investigation to improve the delivery mechanism (Thomas et al., 2003; Ginn et al., 2013). These unwanted effects have led researchers to turn to nonviral vector solutions with high efficiency, stability, and minimal toxicity (Mykhaylyk et al., 2007; Sun et al., 2008). Among them, cationic polymers have been widely studied due to its unique advantages (Merdan et al., 2002). Polyethyleneimine (PEI), a synthetic polymer which has shown high condensation and superior transfection efficiencies, is one of the most used cationic polymers (Wiseman et al., 2003). In order to achieve high efficiency for either DNA or siRNA loading and transfection efficiency, the molecular weight (MW) of the PEI block must be high (e.g., 25 k Da). However, high MW PEI is known to cause systemic toxicity upon intravenous administration (Moghimi et al., 2005). Moreover, another shortcoming is that polycation/DNA or siRNA complexes have the tendency to aggregate when induced by salt- and serum proteins in vivo and can be rapidly cleared by the immune system (Moret et al., 2001). Although PEI can be conjugated with PEG to form block copolymer assemblies and has shown increased stability, reduced toxicity, and lower immunogenicity (Kim et al., 2005; Sato et al., 2007), block copolymers still limit timely release of the internalized gene from the complex due to steric interferences of the associated PEG chains (Grigsby and Leong, 2010; Salcher and Wagner, 2010). Therefore, developing efficient delivery systems based on biomaterials may be an effective means for nucleic acid delivery in therapeutic applications. PEG– PLA micelles are able to resolve parts of the aforementioned problems of gene delivery. In order to systematically deliver DNA expression vectors to tumors, Shukla et al. developed a novel dual nanoparticle (DNP) system to deliver DNA expression vectors to tumor cells. The DNP system was consisted of a DNA expression vector–cationic peptide nanocomplex (NC) surrounded by a PEG–PLA nanoparticle. The results showed that the DNP system could effectively induce apoptosis of tumor cells and possess highly anti-tumor efficacy in vivo (Shukla et al., 2017).

RNA interference (RNAi) is a post-transcriptional gene silencing phenomenon. The discovery of RNAi was through the mechanism of small interfering RNA (siRNA). The mechanism is that siRNA can be incorporated into the RNAinduced silencing complex (RISC) and specifically degrades the target messenger mRNA through complementary base pairing, thereby prohibiting the translation into target proteins (Amjad et al., 2017). Zhao et al. synthesized a biodegradable amphiphilic tri-block copolymer (mPEG2000-PLA3000 b-R15) composed of monomethoxy poly(ethylene glycol), poly(D,L-lactide), and polyarginine. This copolymer can further self-assemble into cationic polymeric nanomicelles for in vivo siRNA delivery. The polymeric nanomicelles showed excellent haemocompatibility and higher cell growth inhibition toward EGFR expressed MCF-7 cells. In vivo experiments further proved the effective tumor growth inhibition effect of the polymeric nanomicelles (Zhao et al., 2012) (**Figure 5**).

In addition to the therapeutic application of genetic material, genes can also be delivered alongside traditional chemotherapy drugs. Zhan et al. investigated the antiglioblastoma effects of RGD-PEG-PEI/pORF-hTRAIL nanoparticle combined with CDX-PEG–PLA-PTX micelle (paclitaxel loaded CDX-poly(ethylene glycol)–block-poly(lactic acid) micelle). When administered the same dosages, the survival time of the intracranial glioblastoma-bearing model mice was significantly longer in the co-delivery (33.5 days) treated group than that of the groups solely treated with CDX-PEG–PLA-PTX (25.5 days), RGD-PEG-PEI/pORFhTRAIL [24.5 days), or physiological saline (21.5 days)]. This research proved the high efficacy for co-delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PTX in the intervention of intracranial glioblastoma by employing tumor-targeted gene carrier RGD-PEG-PEI and brain-targeted micelle CDX-PEG–PLA, respectively (Zhan et al., 2012).

# OTHERS

Curcumin (Cur), a natural polyphenol of Curcuma longa, has been widely researched for its antitumor activities. However, the poor aqueous solubility and low biological availability have limited its further application. Zheng et al. fabricated Curloaded PEG–PLA micelles. The preparation of Cur-MPEG-PLA was very simple and fast. Besides, the micelle group showed a sustained release behavior of Cur and an enhanced toxicity on C6 and U251 glioma cells in vitro. Moreover, compared to free Cur, they induced more apoptosis on C6 glioma cells.

The Cur-loaded micelles also effectively improved the antiglioma activity of Cur in vivo (Zheng et al., 2016) (**Figure 6**). In another study, researchers constructed Cur-loaded pH-sensitive MPEG–PLAPAE micelles. These micelles could shrink from 171.0 nm to 22.6 nm and could increase their surface charge to 24.8 mV, which significantly improved the cell uptake of Cur by MCF-7 cells. Moreover, these micelles also exhibited excellent antitumor efficiency in vivo (Yu et al., 2014). The antitumor activity of Cur-loaded PEG–PLA micelles were also reported in other studies (Yang et al., 2012c; Kumari et al., 2016, 2017).

Antivascular therapy is a distinctive form of cancer treatment. It can cause a selective and rapid shutdown of the tumor vasculature, thus resulting in extensive cancer cell death. Wang et al. developed αvβ3 integrin-targeting peptide (RGD) functionalized polymeric micelles (RFPMs) based on the use of poly(ethylene glycol)-block-poly(D,L-lactide) (PEG–PLA). DOX was conjugated to the PEG–PLA micelle core and combretastatin A4 was physically encapsulated into the micelles (RFPMs-DOX-CA4). The micelles exhibited sequential release kinetics, resulting in sequential killing of endothelial cells and tumor cells in vitro. In B16-F10 tumor-bearing mice, RFPMs-DOX-CA4 showed stronger tumor growth inhibition and significantly higher survival rate compared to other treatment groups (Wang et al., 2011).

# CONCLUSIONS

In the last few decades, polymeric micelles have become one of the most promising nano-delivery systems for the treatment of cancers have been used for the delivery of a variety of cargoes, from conventional chemotherapeutic drugs to specific therapeutic agents and biological macromolecules. Among various polymeric micelles, PEG–PLA based micelles have been intensively studied because of their excellent biodegradability and biocompatibility. Genexol-PM has already been approved for breast cancer treatment in South Korea. With the progressive development of cancer treatment methods, such as photodynamic therapy, photothermal therapy, immunotherapy, and gene therapy, PEG–PLA micelles are being increasingly applied in combination with these treatments. It is worth noting that besides primary passive targeting through the EPR, there is a clear shift toward the utilization of micelles which can be modified for active targeting, controlled delivery of therapeutic agents reliant on the unique tumor microenvironment or external environment, and combination of more than one type of therapeutic payload.

This review has discussed various examples of PEG–PLA micelles being applied in a variety of therapeutic applications for the treatment of cancers. These micelles contain a wide range of modifications including primary modification for passive

Reproduced with permission from Zheng et al. (2016).

targeting, incorporation of targeting ligands, responsiveness to the tumor microenvironment, and the mixing of micelles with drugs and other therapeutic agents. Among them, multifunctional PEG–PLA micelles have gained immense attention due to their versatility in simultaneously incorporating various agents (e.g., chemotherapeutic drugs and RNAi) and their ability to achieve multiple modifications (e.g., active targeting, passive targeting, and response to stimuli) to enhance cancer therapy.

It is promising to be hopeful about the future of PEG–PLA micelles given their inherent advantages. However, it should be noted that the safety of these novel concepts is a major concern. Rollerova et al. validated that PEG-b-PLA NPs might interfere with the activation and function of the hypothalamic-pituitarygonadal (HPG) axis, which might relate to the nanoreprotoxicity of PEG-b-PLA NPs at both the central neuroendocrine and gonadal levels (Rollerova et al., 2015). In addition, PEG-b-PLA NPs also could cause neuroendocrine disrupting effect in the neonatal female rats (Scsukova et al., 2015). Dvoráková et al. indicated a possible age-related association between the oxidative stress and neonatal PEG-b-PLA administration (Dvoráková et al., 2017). Therefore, the PEG–PLA micelles still need to be carefully studied in several animal models and eventually

#### REFERENCES

in human patients.It is known that the simpler the structure of a system, the easier it is to translate. In the future, we should bear this in mind to design more "active" targeting and simple structures of PEG–PLA micellar drug delivery systems. It is promising that PEG–PLA micelles will play an increasingly important role in the wide range of treatment methods against cancer.

# AUTHOR CONTRIBUTIONS

JW and SL produced the first draft. SC and DL revised the manuscript. DL, YH, and JG proposed the outline of the article and revised the draft before submission. CW revised the draft carefully. In additional all authors provided final approval of the manuscript.

#### ACKNOWLEDGMENTS

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 51673190, 51603204, 51303174, 51473165, 51390484, and<PHONE_NUMBER>4) and the Science and Technology Development Program of Jilin Province (Grant Nos.<PHONE_NUMBER>5SF and<PHONE_NUMBER>8SF).

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and in vivo. J. Control. Release 160, 630–636. doi: 10.1016/j.jconrel.2012. 02.022

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Wang, Li, Han, Guan, Chung, Wang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# H2O2-Responsive Nanoparticle Based on the Supramolecular Self-Assemble of Cyclodextrin

#### Zhenqiang Dong1,2† , Yang Kang3† , Qijuan Yuan1 , Manli Luo4 and Zhipeng Gu1 \*

#### Edited by:

Wei Tao, Harvard Medical School, United States

#### Reviewed by:

Peng Miao, Suzhou Institute of Biomedical Engineering and Technology (CAS), China Pedro Fonte, CBIOS – Universidade Lusófona, Portugal Xiaolong Li, Dana–Farber Cancer Institute, United States

\*Correspondence:

Zhipeng Gu<EMAIL_ADDRESS>†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology

Received: 13 March 2018 Accepted: 08 May 2018 Published: 28 May 2018

#### Citation:

Dong Z, Kang Y, Yuan Q, Luo M and Gu Z (2018) H2O2-Responsive Nanoparticle Based on the Supramolecular Self-Assemble of Cyclodextrin. Front. Pharmacol. 9:552. doi: 10.3389/fphar.2018.00552 1 Key Laboratory of Sensing Technology and Biomedical Instrument of Guangdong Province, School of Engineering, Sun Yat-sen University, Guangzhou, China, 2 College of Chemical and Material Engineering, Quzhou University, Quzhou, China, 3 Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China, 4 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China

Designing stimuli responsive, controllable and biocompatible multifunctional nanoparticles is an important progress in the current quest for drug delivery systems. Herein, we devoted to developing a β-cyclodextrin (β-CD) based drug delivery nanoparticles (NPs) that release Bovine serum albumin (BSA) via glucose-responsive gate. The design involves synthesis of sodium alginate with β-CD modified (Alg-β-CD) and methoxypolyethylene glycol (mPEG-Fc) containing ferrocene (Fc) uncharged end-capping. When α-cyclodextrin (α-CD) was added with these two segments, the stable non-covalent supramolecular structure of Alg-β-CD/mPEG-Fc/α-CD can be selfassembled into NPs in aqueous solution. BSA loaded Alg-β-CD/mPEG-Fc/α-CD also has been prepared. Interestingly, these supramolecular Alg-β-CD/mPEG-Fc/α-CD/BSA NPs showed uniform sphere structure and constant BSA loading content. Also, this new kind of NPs can disassemble in the present of hydrogen peroxide (H2O2). Since glucose oxidase (GOD) can oxidize glucose and produce H2O2, so this kind of polymeric NPs can also have glucose responsive behavior in the GOD containing environment. Developed functional Alg-β-CD/mPEG-Fc/α-CD might be a promising drug delivery strategy for diabetes or immunotherapy with more efficiency.

Keywords: responsive, cyclodextrin, self-assemble, nanoparticle, BSA

# INTRODUCTION

For the past decades, stimuli responsive nanoparticles (NPs) based delivery technique have emerged as a promising strategy for controllable drug delivery, especially for protein delivery due to ingenious design (Tao et al., 2013; Wu et al., 2014; Kuang et al., 2016; Ding et al., 2017; Kang et al., 2017b). As smart vehicles, these ideal NPs can undergo reversible physical or chemical changes to control drug release in response to external stimuli such as pH, light, temperature, redox and molecules (Kang et al., 2015; Tao et al., 2016, 2017a,b; Rosenblum et al., 2018; Zhu et al., 2018). For cancer therapy, diverse intracellular and extracellular endogenous stimuli within

FIGURE 1 | Schematic illustration of the formation of Alg-β-CD/mPEG-Fc/α-CD nanoparticles and their enzyme encapsulation and controlled release behavior.

tumor microenvironment, such as low pH value, hypoxia, enzymes and reactive oxygen species (ROS) have been widely investigated in stimuli responsive NPs and explored as the stimuli to selectively trigger drug control release or transfer prodrug systems into active form (Chen et al., 2015; Ruan et al., 2015, 2016; Liao et al., 2016; Wang et al., 2017b).

As an important hallmark, it should be noticed that various studies have demonstrated that hydrogen peroxide (H2O2) could be used as a second messenger in intracellular signaling cascades and would be generated in multiple skin lesions, initiating aging cells and the tumoral tissue (Miao et al., 2015). In this regard, H2O2-responsive NPs would be a promising therapeutic strategy for the treatment of specific disease.

At present, block copolymer approaches for H2O2-responsive NPs has been widely used and rapidly developed. For instance, a tyrosol incorporated copolyoxalate (TPOX) NPs have been developed by Kim et al. (2017), which demonstrated that TPOX NPs released entrapped nile red through the degradation under H2O2 stimulation and could be used as H2O2-responsive therapeutic NPs for the treatment and inflammatory. Recently, Gu group have designed a glucose-responsive mechanism directly utilizing H2O2-sensitive polymeric vesicles by using polyethylene glycol (PEG) and phenylboronic ester (PBE)-conjugated polyserine for smart insulin delivery (Hu et al., 2017). Apart from stimuli-responsive polymeric NPs via cleavable covalent bonds to control their properties, supramolecular based NPs via non-covalent bonds have also been widely studied due to that functional ligands and environmental responsive bonds can be conveniently incorporated through host-guest interaction, π–π interaction, hydrogen bonding interaction and so on (Wang et al., 2015, 2017a). Cyclodextrin (CD), as a most common case of supermolecules, which has been demonstrated that can bind some specific hydrophobic guest molecules into its cavities to construct stable host-guest complex (Yuan et al., 2013). CDs are also considered as a good choice for drug delivery strategy due to their adjustable water solubility, good biocompatibility, and non-toxicity toward biological systems (Kang et al., 2014). In the previous studies, various stimuli-responsive CDs NPs based on host-guest interactions between CDs and different guest molecules [such as azobenzene, ferrocene (Fc), and adamantine] have been widely developed. For instance, Guo and Lei (2015) have developed a kind of redox-responsive cationic supramolecular polymers based on the host-guest interaction between β-CD and Fc, which indicated the potential application for gene nanocarrier. To conduct the redox-stimuli in an effect way, Kang et al. (2017a) have designed a kind of dual redox-responsive micelles which fabricated by methoxypolyethylene glycol conjugated β-CD (mPEG-β-CD) and Fc conjugated camptothecin (Fc-SS-CPT)

segments for achieving dual ROS and glutathione responsive. It is reasonable to design H2O2-sensitive NPs based on CD, which can load and controllable releasing drugs for treatment of specific disease.

In this paper, a novel oxidation responsive rod-coil polymeric NPs have been prepared using water as the self-assemble solvent (**Figure 1**) Firstly, sodium alginate with β-CD end-decoration (Alg-β-CD) and mPEG conjugated with Fc (mPEG-Fc) were designed and synthesized, and then the self-assembly behavior of them in water was studied in detail. This supramolecular copolymer grows up to form comb shaped polymer in aqueous solution by the terminal host-guest inclusion. When adding certain amount of α-CD to this solution, the PEG will form inclusion complex with α-CD to form the rod section while sodium alginate form the coil section. This kind of structure will self-assemble into rod-coil NPs in water and load Bovine serum albumin (BSA). This new kind of NPs can disassemble in the present of H2O2. Since glucose oxidase (GOD) can oxidize glucose and produce H2O2, so this kind of polymeric nanosphere can also have glucose responsive behavior in the GOD containing environment. This will make it very promising to use in biomedical application field in body environment.

# MATERIALS AND METHODS

#### Materials and Reagents

β-cyclodextrin (β-CD), α-Cyclodextrin (α-CD) was purchased from Sigma and dried in a vacuum before use. Sodium alginate (Alg) was purchased from Tianjin Yuanhang Chemicals Co., Ltd., China. Methoxypolyethylene glycol (mPEG, Mw = 2000) was purchased from Shanghai Jingchunshiye Co., Ltd., China. Glucose oxidase was purchased from Aspergillus Niger (GOD, lyophilized powder protein, Mw = 186 000, catalase <4%). BSA was purchased from Aoke Biological Co., Ltd., China. Carboxyferrocene (Fc-COOH), morpholinoethanesulfonic acid (MES), dicyclohexylcarbodiimide (DCC), 4-(dimethylamino) pyridine (DMAP), N-hydroxysuccinimide (NHS), 1-ethyl-3-[3- (dimethylamino)propyl]carbodiimide hydrochloride (EDCI), and other reagents were purchased from Chengdu Kelong Co., Ltd., China.

# Synthesis of Alg-β-CD and mPEG-Fc

Sodium alginate with β-CD was synthesized in two steps with slight modification. Firstly, the mono-6-(p-tolylsulfonyl)-β-cyclodextrin (6-β-CD-OTs) and

mono-6-deoxy-6-hexamethylenediamine-β-CD (6-β-CD-HDA) were synthesized according to the method reported in the previous study (Quan et al., 2010). Briefly, β-CD was grafted onto the sodium alginate backbone via amido link condensation reaction. 1% sodium alginate solution (w/v) was prepared in MES buffer solution (0.1 M) and NaCl (0.5 M), and the pH was adjusted to 6.0. After 29 mg NHS and 95 mg EDC (molar ration of EDC: NHS: COO- = 1: 0.5: 1) were added to 40 mL above alginate solution, the solution was then agitated for 30 min to obtain a homogeneous solution followed by the addition of 10 mL 3.08 % 6-β-CD-HDA MES solution (w/v). The reaction was then continuing at 4◦C for 24 h. Alg-β-CD was obtained after the resulting mixture dialyzed against pure water and then freeze dried. The degree of β-CD substitution (DS) to alginate was calculated according to the followed equation:

$$\frac{I\_1}{I\_2 - 6I\_1 - \frac{2}{7}I\_2} = \frac{7}{4} \text{DS}$$

where I1 is the digital integration of anomeric protons of β-CD, I2 is the total digital integration of 3.4–4.2 ppm. The final degree of substitution (DS) of Alg-β-CD was 25.6 % by this calculated method. 1HNMR (400 MHz, D2O) were shown in **Figure 2**.

mPEG-Fc was synthesized with one pot method. DMAP (0.183 g) and DCC (1.12 g) were added successively to a solution of mPEG (3.0 g) and carboxyferrocene (0.69 g) in dry chloroform (CHCl3, 100 mL) for 24 h at room temperature. After removing dicyclohexylurea (DCU) by filtration, the filtrate was concentrated in a vaccum and purified by dissolving in deionized water and extracting with diethyl ether four times. Subsequently, the product was further purified by using gel column with methanol and water as the solvent to remove the unreacted carboxyferrocene and catalyst residue. Finally, the dark red product was concentrated in a vaccum and dried in the vacuum oven for 2 days. The final DS of PEG-Fc used was 95.4% which was calculated though the integral ratio between Hd and Hf . 1HNMR (400 MHz, CDCl3) were shown in **Figure 3**.

#### Preparation of Alg-β-CD/mPEG-Fc/α-CD and Alg-β-CD/PEG-Fc/α-CD/BSA NPs

PEG-Fc (6.6 mg) and Alg-β-CD (8.4 mg) were added to 3 mL deionized water and kept stirring for 5 h to get a transparent solution. Then α-CD (0.2 g) was added to the above solution and continued to stir over night. For BSA loading, 1.5 mg BSA was added with stirring for 10 min before the addition of α-CD. The solution changes from transparent to turbid gradually indicating the formation of the Alg-β-CD/PEG-Fc/α-CD and Alg-β-CD/PEG-Fc/α-CD/BSA NPs.

### Characterization of the Alg-β-CD/mPEG-Fc/α-CD and Alg-β-CD/mPEG-Fc/α-CD/BSA NPs

The product was characterized by 1H–1H (2D) NOESY NMR spectroscopy which recorded on an Advance Bruker 600 NMR spectrometer. The transmission electron microscopy (TEM)

images were taken by using a JEOL JEM-100CX instrument operating at an accelerating voltage of 80 kV. Each sample were prepared and imaged by Atomic force microscopy (AFM) in tapping mode with a Nanoscope IIIa Digital Instrument. X-ray powder diffraction (XRD) patterns of the samples were recorded by using Cu-Kα irradiation source with X' Pert MPD (20 kV; 35 mA; 2◦ theta/min) to determine the crystalline structures.

#### Self-Assembly NPs Encapsulation and Leakage of BSA

Self-assembly NPs encapsulated BSA were prepared and then their encapsulation efficiency was determined as follows. The BSA loaded NPs (0.5 mg BSA, 0.5%) was centrifuged for 20 min at 12,000 rpm to completely separate the free and encapsulated BSA and the supernatant was analyzed by determined by UV spectrophotometry. The BSA encapsulation efficiency was

FIGURE 6 | X-ray diffraction patterns of (a) α-CD, (b) PEG/α-CD inclusion complex, (c) Alg-β-CD/PEG-Fc/α-CD particles, (d) Alg-β-CD/PEG-Fc.

FIGURE 8 | Bovine serum albumin release from Alg-β-CD/PEG-Fc/α-CD/BSA and Alg-PEG/α-CD/BSA nanoparticles at 37◦C in 12 mL 0.1 M glucose and 10 mg GOD containing solution.

calculated as follows (1):

Bovine serum albumin encapsulation efficiency (%) = mass of BSA used in formulation − mass of free BSA mass of BSA used in formulation × 100

Then the NPs were continuing stored in aqueous solution for 48 h to evaluate leakage of BSA. The mixture was again separated by centrifugation and the supernatant was analyzed for determination of BSA leakage efficiency as above method (2):

Bovine serum albumin leakage efficiency (%) =

$$\frac{\text{mass of BSA in the supernovaantant}}{\text{mass of BSA encapsolated}} \times 100$$

#### In Vitro Release Studies

The Alg-β-CD/PEG-Fc/α-CD self-assembly NPs encapsulated BSA was prepared and centrifuged for in vitro BSA release study (12,000 rpm, 20 min). The lower sediment was added 12 mL 0.1 M glucose solution and 10 mg GOD and kept stirring for 10 min. The solution was placed in a constant temperature incubator and kept the temperature at 37◦C during the whole release process. 2 mL solution had been taken out and centrifugation at 12,000 rpm for 20 min every 6 h. The amount of BSA released in the supernate was collected and determined by UV spectrometry at specified time. The BSA release was quantified when the standard curve was plotted using a standard BSA solution.

#### In Vitro Cell Studies

Methyl tetrazolium (MTT) assay was carried out on murine colon carcinoma cells (CT26) as the cell line for biocompatibility evaluation of the self-assembly NPs. 5 × 103 cells were culture for

24 h to attach in 96-well plates. Then the cells were treated with fresh medium containing 1 mg/mL Alg-β-CD/mPEG-Fc/α-CD NPs and Alg-β-CD/mPEG-Fc/α-CD/BSA NPs. The cell viability was measured after incubation for desired time duration (48 h). The cells without treatment were used as the control. Each group was repeated in triplicate.

Further, confocal laser scanning microscopy (CLSM) was used for monitoring the in vitro endocytosis. Briefly, selfassembly NPs encapsulated Dil were prepared and incubated with CT26 cells at 37◦C for 4 h. After incubation, all samples were stained with LysoTracker Green (Life Technologies, Carlsbad, CA, United States) and 10 µg/mL of Hoechst 33342 (Life Technologies, Carlsbad, CA, United States).

#### Statistical Analysis

All the results obtained in this study were expressed as mean ± standard deviation unless otherwise noted. Comparisons between two groups were made using two-tailed student's t-test and between multiple groups were made using one-way analysis of variance (ANOVA). Statistical analyses were performed using SPSS 21.0 and considered statistically significance when ∗p < 0.05.

#### RESULTS AND DISCUSSION

According to the previous, it has demonstrated that inclusion complexes would form when β-CD interact with Fc group (Tomatsu et al., 2010). Further, the formation of complexation between the Fc groups on mPEG-Fc and β-CD groups on Algβ-CD could be observed by using 2D NMR NOESY spectra. As shown in **Figure 4**, the peaks of β-CD group were in accordance with the resonance of the Fc groups could be found in the 2D NOESY spectrum of Alg-β-CD/PEG-Fc, which indicating that the Fc groups threaded through the cavity of β-CD to form an inclusion complex.

The resulting Alg-β-CD/PEG-Fc was hydrophilic copolymers, which could form transparency solution in water. By adding α-CD to the aqueous solution of Alg-β-CD/PEG-Fc at room temperature, the solution gradually became turbid gradually when the aggregates were formed. The morphology of these aggregates has been observed by TEM and atomic force microscope (AFM). TEM micrographs of **Figure 5a** revealed that the Alg-β-CD/PEG-Fc/α-CD aggregates appeared round line shapes and their diameters were about 100 nm. In the AFM micrographs of **Figure 5c**, Alg-β-CD/PEG-Fc/α-CD selfassembly NPs also showed rounded line shapes with a horizontal length about 100 nm. Particularly, it is noted that most height of the aggregates (<5 nm) was about 20 times smaller than horizontal length. The main reason for such size differences between horizontal and vertical direction might be the collapsing of this NPs. Further, the height of NPs increased about threefold when BSA was encapsulated, which demonstrated that the BSA has been successfully encapsulated into the Alg-β-CD/PEG-Fc/α-CD NPs (**Figures 5b,d**).

The XRD patterns of the α-CD (**Figure 6a**), PEG/α-CD inclusion complex (**Figure 6b**), Alg-β-CD/PEG-Fc/α-CD (**Figure 6c**), and Alg-β-CD/PEG-Fc (**Figure 6d**) has been shown in **Figure 6**. As an important index to evaluate the inclusion structures formation of CD based complex. The pattern of Alg-β-CD/PEG-Fc/α-CD showed in the figure was differs from that of free Alg-β-CD/PEG-Fc or α-CD. It was also found that the homo-PEG crystalline peaks (2 theta = 19.2◦ and 23.3◦ ) and α-CD crystalline peak (2 theta = 21.5◦ ) were absent. According to the previous study, the peak at 2 theta = 19.9◦ is a typical peak of PEG-α-CD channel-type crystallites that demonstrated the inclusion structures of Alg-β-CD/PEG-Fc/α-CD contained PEG-α-CD (Lautens et al., 1990). Meanwhile, the NPs would

form an inner rod-like PEG-α-CD inclusion block surrounded by a protonated coil-like Alg shell when insoluble PEG-α-CD inclusion blocks formed and water became a selective solvent for the Alg-β-CD/PEG-Fc/α-CD. This rod-like block in rodcoil system preferred parallel packing which may result in the formation of hollow NPs promoted by efficient space-filling has been investigated (Jenekhe and Chen, 1999).

As mentioned in the literature and our previous study, the Alg-β-CD/PEG-Fc/α-CD self-assemble NPs were connected by the Fc-β-CD inclusions. These NPs could release BSA through their collapse after the dissociation of Fc-β-CD. To achieve this goal, the oxidization Fc to Fc+ is an efficacy way while β-CD could not form complex with Fc derivatives in their oxidized state (Matsue et al., 1985). To get a clear vision, hydrogen peroxide (H2O2) was chosen as the oxidant in this study. After the addition of H2O2 (1 mL, 30%, H2O2 solution) to Alg-β-CD/PEG-Fc/α-CD hollow NPs (10 mL, 1%), the system became unstable and produced polyrotaxane precipitate in 5 min. The possible mechanism of this phenomenon can be seen in **Figure 7**. The addition of H2O2 dissociated Fc-β-CD inclusion complex and leaving the rod-like hydrophobic PEG-Fc/α-CD part precipitated out of water.

Nowadays, one of the most widely used applications of the stimuli-responsive NPs is in the on demand drug-release field (Tao et al., 2015). It requires the micelle system release the aim drugs gradually to achieve the long-term drug action. Since H2O2 could be produce in a rather sustained way through oxidization of glucose by glucose oxidase (GOD) so that can oxidize Fc to its oxidation state gradually. **Figure 7** shows the UV-vis spectra of the PEG-Fc in 0.1 M glucose before and after 10 mg GOD was added. After adding GOD for 12 h, the absorption peak around 450 nm due to Fc groups d–d transition peak (in reduced form) almost disappeared indicating that the Fc groups in the PEG-Fc could be oxidized to ferrocenium as anticipated (Yamamoto et al., 2000). Tan et al. (2012) have been developed the glucose sensitive hydrogel using the GOD and glucose interaction to produce hydrogen peroxide to disintegrate the ICs of Fc and β-CD. Therefore, it is possible to get glucose sensitive Alg-β-CD/PEG-Fc/α-CD micelles by adding GOD to the Alg-β-CD/PEG-Fc/α-CD system.

Otherwise, serum albumins, as a major soluble protein, have got much attention and been widely used due to their physiological functions. It has been applied in the nanomedicine field because of they serve as a depot protein and as a transport protein for a variety of compounds. BSA has been one of the most extensively member used as a protein drug model, particularly because of its structural homology with human serum albumin (Papadopoulou et al., 2005). The incorporation of BSA into Alg-β-CD/PEG-Fc/α-CD particles had been studied. The encapsulation efficiency and leakage efficiency was 71.4 and 6.6%, respectively. The in vitro release profile of BSA from Algβ-CD/PEG-Fc/α-CD/BSA and Alg-PEG/α-CD/BSA particles was presented in **Figure 8**. In Alg-β-CD/PEG-Fc/α-CD/BSA particles group, the BSA appeared to be released in a continuous way. After 48 h, about 80% of BSA had been released. While as a counterpart, the Alg-PEG/α-CD/BSA group (BSA encapsulation efficiency was 66.7%), under the same condition, there was less than 20% of BSA had been released which is much less than the Alg-β-CD/PEG-Fc/α-CD/BSA group. This result further confirmed that in glucose and GOD containing environment the Alg-β-CD/PEG-Fc/α-CD/BSA particle achieved BSA delayed release behavior through the β-CD-Fc inclusion complex as redox trigger.

The biocompatibility of NPs is of critical importance to their eventual success (Tao et al., 2014). As shown in **Figure 9A**, the results demonstrate the non-toxic of Alg-β-CD/PEG-Fc/α-CD and Alg-β-CD/PEG-Fc/α-CD/BSA NPs, which could be used as the biocompatible drug delivery system. The performance of NPs cellular internalization through the observation of NPs encapsulated Dil interaction with labeling CT26 cells (**Figure 9B**). The results indicated that large numbers of Alg-β-CD/PEG-Fc/α-CD NPs entered the cells after 4 h and some of them began to enter into cell nucleus.

# CONCLUSION

In summary, Alg-β-CD/PEG-Fc/α-CD rod-coil hollow NPs have been prepared through the supramolecular self-assemble. The rod-part PEG-α-CD and the coil-part Alg were connected through β-CD-Fc inclusion complex. The BSA enzyme was successfully encapsulated in Alg-β-CD/PEG-Fc/α-CD hollow sphere in aqueous solution. These Alg-β-CD/PEG-Fc/α-CD hollow NPs showed good H2O2 sensitivity. Further study showed that this nanoparticle encapsuled BSA can achieve BSA delayed drug-release behavior in the glucose containing environment at the existence of GOD. Therefore, this study exhibits that using the host-guest interactions would be a general, useful and effective way for fabricating stimuli-responsive drug delivery NPs. Furthermore, the glucose mediated H2O2 sensitive behavior of the Alg-β-CD/PEG-Fc/α-CD NPs may benefit its application in more and more fields such as enzyme immobilization, drug delivery and micro-reactors.

# AUTHOR CONTRIBUTIONS

ZG designed experiments, supervised the experiments, and revised and finalized the manuscript. ZD and YK performed the experiments, analyzed the data, and prepared the figures and the manuscript. QY performed the cell culture. ML contributed to the results discussion and paper writing. All authors reviewed and approved the final paper.

# FUNDING

This study was supported by the funding and generous support of the National Natural Science Foundation of China (21704104), West Light Foundation of the Chinese Academy of Sciences (2016XBZG\_XBQNXZ\_B\_003). Meanwhile, we also want to sincerely acknowledge the funding from the Guangdong Province Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation (2017B030314026).

### REFERENCES

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the π-conjugated main chain. preparation, optical properties, redox behavior, and mössbauer spectroscopic analysis. Macromolecules 30, 5390–5396. doi: 10.1021/ma9704368

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Dong, Kang, Yuan, Luo and Gu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Icariin Activates Autophagy via Down-Regulation of the NF-κB Signaling-Mediated Apoptosis in Chondrocytes

Bobin Mi1 , Junqing Wang2 , Yi Liu1 , Jing Liu1 , Liangcong Hu1 , Adriana C. Panayi3 , Guohui Liu1 \* and Wu Zhou1 \*

1 Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 2 State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, China, 3 Addenbrooke's Hospital, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom

#### Edited by:

Wei Tao, Harvard Medical School, United States

#### Reviewed by:

Fang Chen, Harvard Medical School, United States Chun Yao, Harvard University, United States

#### \*Correspondence:

Guohui Liu<EMAIL_ADDRESS>Wu Zhou<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology

Received: 03 April 2018 Accepted: 21 May 2018 Published: 06 June 2018

#### Citation:

Mi B, Wang J, Liu Y, Liu J, Hu L, Panayi AC, Liu G and Zhou W (2018) Icariin Activates Autophagy via Down-Regulation of the NF-κB Signaling-Mediated Apoptosis in Chondrocytes. Front. Pharmacol. 9:605. doi: 10.3389/fphar.2018.00605 Osteoarthritis (OA) is a common chronic and degenerative joint condition that is mainly characterized by cartilage degradation, osteophyte formation, and joint stiffness. The NF-κB signaling pathway in inflammation, autophagy, and apoptosis plays a prominent role in the progression of OA. Icariin, a prenylated flavonol glycoside extracted from Epimedium, have been proven to exert anti-osteoporotic and anti-inflammatory effects in OA. However, the action mechanisms of its effect on chondrocytes have yet to be elucidated. In the present study, we demonstrated that the in vitro therapeutic effects of icariin on rat chondrocytes in a dose-dependent manner. We found that TNF-α induced the production of IL-1, IL-6, IL-12, reactive oxygen species (ROS), nitric oxide (NO), Caspase-3, and Caspase-9 in chondrocytes. We also provided evidence that TNF-α inhibited autophagy markers (Atg 5, Atg 7) and prevented LC3 I translate to LC3 II. Furthermore, TNF-α induced matrix metalloproteinase (MMP)3 and MMP9 expression. The negative effects of TNF-α on chondrocytes can be partially blocked by treating with icariin or ammonium pyrrolidinedithiocarbamate (PDTC, an NF-κB inhibitor). The present study data also suggested that icariin suppressed both TNF-α-stimulated p65 nuclear translocation and IκBα protein degradation. These results indicated that icariin protected against OA by suppressing inflammatory cytokines and apoptosis, through activation of autophagy via NF-κB inhibition. In conclusion, icariin appears to favorably modulate autophagy and apoptosis in chondrocytes making it a promising compound for cartilage tissue engineering in the treatment of OA.

Keywords: icariin, tissue engineering, autophagy, apoptosis, chondrocytes

# INTRODUCTION

Osteoarthritis (OA) is a chronic degenerative joint disease that occurs mostly in the elderly (Glyn-Jones et al., 2015). As the population ages, the occurrence of OA increases and, consequently, finding an effective treatment is imperative. The capacity of chondrocytes to regenerate normal cartilage matrix architecture declines with aging, resulting in cartilage degradation and erosion

(Blanco et al., 2011). In the cases of OA, various changes occur in the cartilage including inflammation, oxidative stress, loss of cartilage matrix, autophagy, and apoptosis.

During the progression of OA, the increased expression of inflammatory cytokines (such as TNF-α and IL-1β) in articular cartilage and synovium contribute to the degradation and erosion of cartilage (Raman et al., 2018). At the same time, autophagy and apoptosis occur in the progression of OA. Autophagy is necessary for maintaining the cell's metabolism and homeostasis, and for cellular quality control by clearing waste or damaged proteins and organelles (Galluzzi et al., 2014). The dysregulation of autophagy that happens with OA contributes to the degeneration of the articular cartilage (Vasheghani et al., 2015). The importance of autophagy in preventing age-related OA has been demonstrated by the increasing number of studies on the topic (Duarte, 2015; Loeser et al., 2016). In addition, more and more evidences suggest that increased chondrocyte apoptosis induces cartilage degeneration in OA (Kobayashi et al., 2016). Thus, activation of autophagy and inhibition of apoptosis in chondrocytes may limit OA progress.

Recently, various biomaterials, such as hydrogels, have been used as drug delivery systems to regulate chondrocyte autophagy (Chen et al., 2016). Driven by the rapid progression of nanomedicine and nanotechnology (Tao et al., 2015, 2017a; Ding et al., 2017; Rosenblum et al., 2018), compounds have been increasingly studied in the context of regeneration medicine and tissue engineering. However, few compounds are reported to be useful for tissue engineering in cartilage repair. Icariin is a well-known compound extracted from Herba Epimedil, with a wide range of pharmacological effects, including antiinflammatory, anti-atherosclerotic, and anti-oxidative properties (Fang and Zhang, 2017; Wang G.Q. et al., 2017; Xiong et al., 2017). Recently, icariin-mediated chondroprotective effects have attracted growing attention. Icariin protects against OA by inhibiting overexpression of metalloproteinase 13 (MMP-13) and proinflammatory cytokines in chondrocytes (Zeng et al., 2014). In addition to inhibiting H2O2-induced human umbilical vein endothelial cell apoptosis, icariin suppresses NF-κB signaling in macrophages (Wang and Huang, 2005; Chen et al., 2010). The effect of icariin on chondrocyte autophagy and apoptosis, however, remains unclear.

In this study, we investigate whether icariin has chondroprotective effects against TNF-α-induced cell death. These effects might be closely related with autophagy activation and apoptosis inhibition. In addition, we explore the underlying mechanisms of icariin-mediated cell autophagy and apoptosis.

# MATERIALS AND METHODS

#### Reagent, Antibodies, and Ethics Statement

Icariin was purchased from Sigma-Aldrich (MO, United States). Cell Counting Kit-8 (CCK8) was purchased from MedChemExpress (NJ, United States). Antibodies against ATG5, ATG7, LC3, p65, phosphorylated p65 (p-p65), IκBα, MMP3, MMP9 were purchased from Abcam (Cambridge, United Kingdom). Antibody against GAPDH was purchased from Cell Signaling Technology, Inc. (MA, United States). The NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) was purchased from Abcam (Cambridge, United Kingdom). Enzymelinked immunosorbent assay (ELISA) kits were purchased from Bio-Swamp Life Science (Shanghai, China). The Sprague-Dawley (SD) rat was purchased from The Center of Experimental Animal, Tongji Medical College, Huazhong University of Science and Technology. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology.

#### Cell Isolation and Culture

Chondrocytes were isolated from the articular cartilage of 7–10 day-old male SD rats. Briefly, pieces of articular cartilage were digested with 0.25 mg/mL trypsin for 30 min and 2 mg/mL collagenase type II for 8 h at 37◦C. After digestion, isolated chondrocytes were passed through a 180-µm filter and the cells were centrifuged and washed with PBS several times. The cells were then isolated and stained with trypan blue to evaluate cell viability. Chondrocytes with viability greater than 85% were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 IU/ml penicillin, 100 µg/ml streptomycin at 37◦C, 5% CO2. When the cells were treated with 50 ng/ml TNF-α, 10 µM icariin, 10 µM PDTC, icariin or PDTC were added to the medium 2 h prior to TNF-α addition. In the autophagic flux assay, 0.1 µm bafilomycin A1 was added to the medium 1 h prior to icariin or PDTC addition. In the experiments, chondrocytes were treated with PBS (control), TNF-α, icariin with TNF-α, or PDTC with TNF-α for 24 h. All experiments were conducted in triplicate.

# Cytotoxicity Assay

Rat chondrocytes were seeded in 96-well plates (5 × 103 cells/well) overnight, followed by treatment with various concentrations of icariin for 24 h, 48 h, and 72 h at 37◦C in an atmosphere containing 5% CO2. Following this, 10 µl CCK8 solution was added to each well and the cells were cultured at 37◦C for 2 h. The OD value was then measured with a microplate reader (Thermo Fisher Scientific, United States) at 450 nm.

# ELISA

Chondrocytes were seeded onto a 24-well plate at 2 × 104 cells per well. Following 24 h incubation, cells were treated with PBS, TNFα, icariin with TNF-α, or PDTC with TNF-α. The supernatants were collected after 24 h incubation and the levels of IL-1, IL-6, and IL-12 were quantified using the ELISA kits.

#### Western Blot

Total proteins were extracted using cold RIPA buffer containing protease inhibitor (Boster Biological Technology, Ltd., Wuhan, China). Proteins were quantified using the bicinchoninic acid protein assay kit (BCA kit) according to the manufacturer's

instructions. Equal amount of proteins (10 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, United States). Membranes were blocked in 5% bull serum albumin (BSA, Sigma, United States) in TBST for 2 h, then incubated with primary antibody at 4 ◦C overnight. The primary antibodies were as follows: p65 (1:2000), p-p65 (1:2000), IκBα (1:5000), ATG5 (1:5000), ATG7 (1:10000), LC3 (1:3000), MMP3 (1:10000), MMP9 (1:10000), and GAPDH (1:1000). After washing with TBST (50 mM Tris PH 8.0, 150 mM NaCl, 0.01% Tween-20) three times, the membrane was incubated with HRP-conjugated corresponding secondary antibodies (Goat Anti-Rabbit IgG, 1:10000) for 1 h at room temperature. Following washing, the immunoreactive proteins were visualized with the ECL western detection kit (Thermo Fisher Scientific, United States). The band density was quantified using TANON GIS software (Tanon, Shanghai, China).

#### Reverse Transcription-Quantitative (RT-q) PCR

Total RNA from chondrocytes was extracted using Trizol reagent (Thermo Fisher Scientific, United States). Total RNA was reverse-transcribed to cDNA with RT Master Mix (Takara Japan). The RT-PCR was performed with SYBR Master Mix using StepOne-Plus system (ABI, United States) under the following conditions: denature at 95◦C for 30 s, anneal at 60◦C for 1 min and extend at 95◦C for 5 s. The gene expression was analyzed by 2−11Ct method using GAPDH as the internal control. The primer sequences were as follows: ATG5, forward (50 -AA CGAGAAGCAGAGCCA-30 ) and reverse (50 -ATGCCAT TTCAGGGGTG-30 ); ATG7, forward (50 -GAAGAACCAGAAA GGAGG-30 ) and reverse (50 -CAGGCACTTGACAGACAC-30 ); Bax, forward (50 -TGGTTGCCCTCTTCTA-30 ) and reverse (50CACCCTGGTCTTGGAT-30 ); Bcl-2 forward (50 -CACAG AGGGGCTACGAGT-30 ) and reverse (50 -CAGGCTGGAAGG AGAAGA-30 ); GAPDH, forward (50 -CAAGTTCAACGGCA CAG-30 ) and reverse (50 -CCAGTAGACTC CACGAC AT-30 ).

#### Transmission Electron Microscopy (TEM)

Collected cells were fixed with 2.5% glutaraldehyde for 24 h, which followed by fixed with 1% osmium tetroxide for 1 h at 4 ◦C. After dehydrated with a series of ethanol concentrations (50, 70, 80, 90, and 100%) for 10-min intervals, the samples were incubated in a mixture of acetone and epoxy resin (v:v = 1:1) for 6 h, followed by incubation with pure epoxy resin for 4 h. After semi-thin sectioning, cells were stained with 0.5% toluidine blue and observed under the microscope. Finally, the ultra-section sections were observed using a TEM (Hitachi, Japan).

# Cell Cycle Assay

Collected cells were washed and suspended in 0.3 mL PBS containing 10% FBS and 0.7 mL ethyl alcohol for 24 h at −20◦C. The cells were then suspended with 0.1 mL RNase A (1 mg/mL) and 0.4 mL propidium iodide (PI) (50 µg/mL) for 10 min. The percentage of cells in the different stages was measured using Flow Cytometry.

# Cell Apoptosis Assay

Collected cells were stained with a mixture of calcein-AM and PI solution for 20 min. Fluorescence images of cells were then recorded using an inverted fluorescent microscope. The percentages of cell death were evaluated by calculating the number of PI-stained (dead, red) and calcein-AM-stained (live, green) cells. The number of cells were counted in five random fields by three independent authors. The mean value of each measurement was used for analysis.

# Measurement of ROS Production