Docetaxel induces apoptosis in KB cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo

Accepted Manuscript Folate acid-Cyclodextrin/Docetaxel induces apoptosis in KB cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo

Jin Tao, Jiaojiao Xu, Fangcheng Chen, Beihua Xu, Jianqing Gao, Ying Hu PII: DOI: Reference:

S0928-0987(17)30614-0 doi:10.1016/j.ejps.2017.10.039 PHASCI 4285

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

26 July 2017 28 October 2017 30 October 2017

Please cite this article as: Jin Tao, Jiaojiao Xu, Fangcheng Chen, Beihua Xu, Jianqing Gao, Ying Hu , Folate acid-Cyclodextrin/Docetaxel induces apoptosis in KB cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2017.10.039

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ACCEPTED MANUSCRIPT Folate acid-Cyclodextrin / Docetaxel induces apoptosis in KB cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo Jin Taoa, #, Jiaojiao Xua, #, Fangcheng Chena, Beihua Xua, Jianqing Gaob,

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Ying Hua*

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a. Zhejiang Pharmaceutical College, Ningbo, Zhejiang, China

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b. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China

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Corresponding author:

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Prof. Ying Hu

Zhejiang Pharmaceutical College

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No. 888, East section, Yinxian Main Road, The Zone of Higher Education, Ningbo, Zhejiang, 315100, China.

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Tel: +86 574 8822 2707; Fax: +86 574 8822 3023 E-mail: [email protected] Contributed equally to the work of this manuscript.

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Keywords: DTX/FA-CD inclusion complex, mitochondrial, apoptosis, cancer therapy

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1. Background Docetaxel (DTX) is a potent antitumor drug that has been used to treat prostate, breast, and ovarian cancer and is more effective compared with paclitaxel(Lee et al., 2009;

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Wang et al., 2012). However, similar to most antitumor drugs, DTX has a low solubility in water and low targeting specificity, which lead to side effects and low

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bioavailability(Ferrati et al., 2015; Hami et al., 2014). Several research efforts have attempted to improve the solubility issues through the use of nano-drug delivery

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systems, such as liposomes and micelles, as vectors(Cao et al., 2015; Liang et al., 2015). Cyclodextrins have been demonstrated as excellent materials to increase drug

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solubility by inclusion complexes (Ferrati et al., 2015; Mura, 2014). To further improve drug targeting, antibodies(Geng et al., 2016) or peptides(Velascoaguirre et al.,

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2015) with specific binding site in tumor cells or tissues are modified on the vector surface. Folate receptors (FRs) are generally overexpressed on the surface of cancer

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cells(Parker et al., 2005), and thus serve as a potential target for cancer targeting

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therapeutics. Some researchers have grafted folic acid onto the surface of the nanoparticles, which could significantly improve drug accumulation in tumors, to

2016).

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enhance treatment efficiency and reduce side effects (Butt et al., 2016; Li et al.,

Clarifying the precise mechanism of cancer drugs is a key to further improvement of

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tumor therapies. Antitumor drugs also can cause chromosome aggregation and DNA degradation and induce cancer cell apoptosis, which make programmed cell death and inhabit tumor growth, as a new potential and effective antitumor targeted therapy. Apoptosis is initiated either through the intrinsic mitochondrial pathway or the extrinsic pathway(Ma and Yang, 2016). Most anticancer drugs activate apoptosis via the intrinsic mitochondrial pathway, which involves affecting the mitochondrial membrane potential, generation of reactive oxidative species (ROS), and the expression of apoptosis-related proteins(Giampazolias and Tait, 2015; Park et al., 2017). 3

ACCEPTED MANUSCRIPT We previously constructed DTX/folate acid-cyclodextrin (FA-CD) inclusion complexes and demonstrated that these complexes kill tumor cells may by inducing apoptosis. Our cellular uptake analyses suggested that these complexes had strong affinity to cancer cells with high FR expression(Xu et al., 2016a; Xu et al., 2016b). In the present study, we further investigated the molecular mechanism of the apoptotic

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effect of DTX/ FA-CD inclusion complexes against human oral squamous carcinoma

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KB cells and its antitumor effects in vivo (Scheme 1).

2.1 Materials

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2. Materials and methods

6-Deoxy-6-[(2-aminoethyl) amino]-β-cyclodextrin (CDEn) was purchased from FA-CD was synthesized by our lab.

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Zhiyuan Biotechnology (Shandong, China),

DTX was purchased from Meilunbio (Dalian, China). The Reactive Oxygen Species

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Assay Kit, Mitochondrial Membrane Potential Assay kit with JC-1, glutathione (GSH) assay kit, and Catalase Assay Kit were purchased from Beyotime Institute of

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Biotechnology (Zhejiang, China). Cy5.5 NHS ester (Cy5.5, water-insoluble red

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fluorescent dye) was purchased from Shanghai XiBao Biological Technology (Shanghai, China). Ki-67 assay kit was purchased from Maxim (Fuzhou, China). 2.2 Preparation of DTX/FA-CD and Cy5.5/FA-CD inclusion complexes

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DTX/FA-CD inclusion complexes were prepared by a simple suspension method as previously described(Xu et al., 2016a). Briefly, solid DTX was added to FA-CD

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aqueous solution in a 1:1 mol equivalent proportion and the solution was ultrasonicated at 25°C using an ultrasonic system at 90% amplification (1000 W) for 4 h. The suspension was then centrifuged for 10 min at 10,000 ×g and passed through a 0.45 µm millipore membrane filter and then dried in a refrigerator to produce the solid products. Cy5.5/FA-CD inclusion complexes were prepared like DTX/FA-CD. 2.3 Cell culture KB cells, a human oral squamous carcinoma cell line from the Shanghai Institute for Biological Sciences (Shanghai, China), were cultured in RPMI-1640 containing 10% 4

ACCEPTED MANUSCRIPT fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37˚C with 5% CO2. 2.4 Measurement of ROS Cells were treated with 24 μM DTX/FA-CD inclusion complexes and different concentrations of DTX for 24 h. Cells were harvested and analyzed on a flow cytometer (C6, Becton Dickinson, US) with the Reactive Oxygen Species Assay Kit

2.5 Mitochondrial membrane potential (ΔΨm) assessment

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according to the manufacturer’s instructions.

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Mitochondrial membrane potential was evaluated using the assay kit according to the manufacturer’s instructions. Cells were treated with 24 μM DTX/FA-CD inclusion

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complexes and different concentrations of DTX for 24 h. Cells were then harvested and incubated with JC-1. Samples were analyzed by flow cytometry with settings of

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FL1 (green, FITC) at 530 nm and FL2 (red, PE) at 590 nm. 2.6 Measurement of GSH

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GSH activity was evaluated by the GSH assay kit according to the manufacturer’s instructions. Briefly, following treatment, cells were washed and fluorescence

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intensity was determined at 412 nm using a microplate reader (Multiskan, Thermo,

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US). 2.7 Catalase activity assay

Catalase level was evaluated by the Catalase Assay Kit according to the

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manufacturer’s instructions. Briefly, after treatment, cells were lysed and total proteins were quantified by BCA analysis (Beyotime, China). Catalase activity was

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measured at 520 nm using a microplate reader. 2.8 Western blot analysis After drugs treatment, total protein was extracted from cells and quantified by BCA analysis. Proteins were separated by electrophoresis in SDS-PAGE and transferred to PVDF membranes. After blocking, membranes were incubated with primary antibodies at 4 ℃ for overnight and secondary antibodies, finally image using a chemiluminescence imaging system and analyzed by ImageJ software. 2.9 In vivo biodistribution study Four to six week-old male BALB/c nude mice were housed in specific pathogen-free 5

ACCEPTED MANUSCRIPT animal facility. All animal experiments were conducted according to the guidelines and approved by the ethic committee of Zhejiang Pharmaceutical College. Approximately 1×106 KB cells were inoculated subcutaneously into the left armpit of male BALB/c nude mice. When the tumor size reached around 150 mm3, mice were intravenously injected with free Cy5.5, Cy5.5/CD, or Cy5.5/FA-CD at a dose of 12

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mg/kg, respectively. Fluorescence imaging experiments were performed at 1, 5 7, 12 and 24 h post-injection using an in vivo imaging system FX Pro (Kodak, USA)

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equipped with an excitation band pass filter at 630 nm and an emission at 700 nm. After living imaging, mice were sacrificed and tissues were collected followed by

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imaging using the same imaging system. 2.10 In vivo antitumor activity

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2.10.1 Tumor growth inhibition

To construct a xenograft tumor model, KB cells (1×106) were subcutaneously injected

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into the left armpit of male BALB/c nude mice. When the tumor size reached around 150 mm3, the mice were randomly divided into four groups (with 10 nude

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mice/group), which were treated with PBS, Taxotere® (12 mg DTX/kg), DTX/CD (12

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mg DTX/kg) or DTX/FA-CD (12 mg DTX/kg) by intravenous injection once every six day for five times. Tumor growth was measured every seven day using digital calipers. The tumor volume was calculated as follows: tumor volume=(major

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axis)×(minor axis)2/2. The body weight and surviv.al curve were also monitored. Thirty-four days after the final injection, tumors were harvested and subjected to

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various tests.

2.10.2 Histological study After the end point of experiment, the tumors were fixed for 24 h in 10% paraformaldehyde, and were dissected for histological study using hematoxylin and eosin (H&E) staining. The immune histochemical staining of tumors was conducted with antigen Ki-67. The results were analyzed by digital microscope (MC 170 HD, Leica, Gemany). 2.11 Statistical analysis All data are expressed as mean±SD for three times and statistically analyzed by t-test 6

ACCEPTED MANUSCRIPT or one-way analysis of variance (ANOVA) using GraphPad Prism 7 software. P<0.05 was considered as statistically significant.

3. Results 3.1 Induced ROS generation

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To examine the cytotoxic mechanism of DTX/FA-CD complexes, we evaluated effects on the levels of ROS, which is a typical characteristic of intrinsic

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apoptosis(Yang et al., 2016), in KB cells. KB cells were treated with equal concentrations (24 μM) of Taxotere, DTX/CD, or DTX/FA-CD for 12 h and then

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ROS levels were determined by flow cytometry. Results showed that the DTX induced a remarkable increase of ROS production (Figure 1A). We next evaluated

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ROS production by treated with different time of DTX/FA-CD complex and found that 24 μM DTX induced an increase of ROS production in a time-dependent manner

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(Figure 1B). Interestingly, ROS levels in DTX/FA-CD complex-treated cells at 0.5 h and 1 h were significantly lower than controls (P<0.001). Moreover, the concentration

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of DTX/FA-CD complex and the level of ROS have concentration-independent

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(Figure 1C).

3.2 Assessment of ΔΨm

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Mitochondria play a significant role in apoptosis of certain cancer cell lines(Caino and Altieri, 2016). The ΔΨm is generated by proton transmembrane transport into the

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matrix through the proton pump of the mitochondrial inner membrane. During apoptosis under physiological conditions, mitochondria of apoptotic cells exhibit a low ΔΨm(Zhang et al., 2016). To further examine the mechanism of apoptosis caused by the DTX/FA-CD complex, we evaluated the ΔΨm using fluorescent probe JC-1 by flow cytometry. KB cells were treated with Taxotere, DTX/CD, and DTX/FA-CD for 12 h, the ratio of red to green all have a significance decrease, especially DTX/FA-CD (Figure 2A). Cells were treated with 24 μM DTX/FA-CD for different times and the ΔΨm was remarkably decreased at 1 h and 12 h. (Figure 2B). We did not detect any significant differences in cells treated with various concentrations of DTX/FA-CD for 7

ACCEPTED MANUSCRIPT 12 h (Figure 2C). Together these findings suggest that DTX/FA-CD induces loss of ΔΨm and early apoptosis in a time- and concentration-independent manner.

3.3 Depletion of GSH GSH is important intracellular thiol-containing oxide redox regulation system. The

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decrease of GSH is a protential signal of early apoptosis (Liu et al., 2014). Our results showed that DTX/FA-CD could upregulate ROS in KB cells, and indicate it may

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induce changes in GSH. KB cells were treated with Taxotere, DTX/CD, and DTX/FA-CD for 12 h, and the levels of GSH were all decreased compared with

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controls (Figure 3A). The levels of GSH were opposite to ROS production. Furthermore, with the increase of incubation time and concentration of DTX/FA-CD,

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the level of GSH also gradually decreased (Figure 3B and 3C). This suggests that

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DTX/FA-CD can reduce GSH level in a time- and concentration-dependent manner.

3.4 Assessment of catalase

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Catalase, which is used to study the role of ROS in gene expression and apoptosis,

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can catalyze the decomposition of hydrogen peroxide(Scheit and Bauer, 2014). Overproduction of catalase is a typical characteristics of apoptosis(BAUER and MOTZ, 2016; Scheit and Bauer, 2014). We found a considerable increase of catalase

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level in DTX-treated cells (Figure 4). The levels of catalase were remarkably increased in KB cells treated with different dosages. Further study showed that

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DTX/FA-CD could increase the catalase level in a time- and concentration-dependent manner.

3.5 Assessment of mitochondrial dependent apoptosis protein expressions It is known that mitochondrial potential change is a crucial stage in drug-induced apoptosis and also resulted some protein expression changing which play key roles in mitochondrial-mediated. We treated KB cells with DTX/FA-CD and using western blot to assess the protein expression. There was a significant increase in the expression of pro-apoptotic proteins BAX, caspase-3, caspase-9 and p53, where as 8

ACCEPTED MANUSCRIPT anti-apoptotic protein Bcl-2 showed a significant decrease after KB cells were treated with DTX/FA-CD (Figure 5). And also shown the expression of related proteins were time-dependent and concentration-dependent after treat with DTX/FA-CD (Figure 5B and 5C). Based on the above results, DTX/FA-CD have potential role in inducing

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apoptosis via mitochondrial apoptotic pathway.

3.6 In vivo and ex vivo imaging

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To investigate the distribution of DTX/FA-CD complex in vivo, we used fluorescent dye Cy5.5 to instead DTX, which have a similar molecular weight and lipid solubility

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of DTX, and injected free Cy5.5, Cy5.5/CD or Cy5.5/FA-CD at a dose of 12 mg/kg via the tail vein in KB cell-bearing nude mice. As shown in Figure 6A, the

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fluorescence intensity was increased in tumors after administration of Cy5.5/CD and Cy5.5/FA-CD compared with free Cy5.5. In addition, in the Cy5.5/CD group, Cy5.5

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began to distribute in para-carcinoma tissue, while Cy5.5 also accumulated in tumors in the Cy5.5/FA-CD group. We also performed imaging of fluorescence intensity of

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tumors and major organs after administration. After 48 h post-administration, the

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order of fluorescence intensity levels in tumor tissues was as follows: Cy5.5/FA-CD > Cy5.5/CD > Cy5.5 (Figure 6B). As shown in Figure 6C, fluorescence intensity was detected at 1, 12, 24 and 48 h after administration of Cy5.5/FA-CD. Cy5.5 quickly

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accumulated in tumors at 1 h after injection and was detectable up until 48 h. Obviously, the fluorescence intensity had weak signal in other organ. In other words,

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drug was less accumulated in other organ that similar to we used HPLC analysis(Xu et al., 2016b). These results suggest that Cy5.5/FA-CD showed the ability to target tumors compared with Cy5.5/CD and free Cy5.5.

3.7 In vivo tumor growth suppression In previous study showed that the DTX/FA-CD complex can suppresses KB cell proliferation and induces apoptosis of KB cells(Xu et al., 2016b). To investigate the potential of this delivery system in cancer therapy, we tested the anti-tumor growth effect of DTX/FA-CD in a murine KB solid tumor model. As shown in Figure 7A and 9

ACCEPTED MANUSCRIPT 7B, tumor size in the mice receiving the FA-modified inclusion complexes treatment was the smallest among all the tested groups. At day 34, the tumor volume in mice treated with DTX/FA-CD complex was around 20% of the PBS group, and the average tumor volume of the DTX/ CD group was similar to the Taxotere groups. This indicated that DTX mediated by FA-CD can significantly improve the capable of

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KB tumor growth suppression. Notably, no significant changes were detected among the body weight of PBS,

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DTX/CD and DTX/FA-CD groups (Figure 7C), indicating that the DTX/FA-CD complex showed low toxicity in vivo. The tumor weights were measured after 34 day

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(Figure 7D). In contrast to an inhibition rate of 59.3% in the DTX/CD group and 53.1% in the Taxotere group, the DTX/FA-CD exhibited an enhanced efficiency with

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86.0% (Figure 7F).

H&E staining revealed the necrosis in tumors after treatment with drugs. As shown in

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Figure 7G, treatment with DTX/FA-CD complex resulted in a remarkable decrease in the number of cancerous cells compared with control groups. Ki-67 expression is a

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well-established marker of cell proliferation(El-Naa et al., 2016) and also plays a key

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role in cancer development and metastasis(Xu et al., 2016c). Figure 7H presents the histological and immunohistochemical analyses in the tumor tissue after treatment. Taxotore, CD/DTX and DTX/FA-CD groups showed low expression of Ki-67,

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especially the DTX/FA-CD group, compared with the PBS group. This suggests that

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DTX can suppress the expression of Ki-67 in tumor tissues.

4. Discussion

DTX can increase the polymerization of tubulin and inhibit microtubule depolymerization, which leads to the formation of stable non-functional microtubules. DTX exhibits excellent antitumor activity by disruption of cancer cell mitosis, which causes formation of abnormal microtubules and a G2/M phase block(Blagosklonny and Fojo, 1999; Wang et al., 2012). Although DTX has a significant antitumor effect, it is also associated with side effects, which restricts its application(Liu et al., 2008). Cyclodextrin materials, which have a hydrophilic shell and hydrophobic cavity, 10

ACCEPTED MANUSCRIPT increase the water solubility, stability and biocompatibility of drugs by formation of inclusion complexes with hydrophobic drugs(Chen et al., 2016; Ferrati et al., 2015). Ferrati(Ferrati et al., 2015) prepared hydroxypropyl β-cyclodextrin/DTX inclusion complexes, which increase the water soluble of DTX and prolonging release time, also showed good antitumor effect. However, some research had suggested that the

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inclusion complexes was limited application in clinical. In one sides, the inclusion complexes were easily release drug to blood due to the weak bonding force of

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cyclodextrin and drug which result the blood component will replacement drugs(Kurkov et al.; Mccormack and Gregoriadis, 1998). On the other sides, the

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inclusion complexes had rarely active target in vivo(P et al., 2015). In previous study, we had prepared the DTX/FA-CD inclusion complexes with high stability constant, so

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the DTX/FA-CD was stable in the blood. To further improve targeting in vivo, many researchers have modified the carrier surfaces of nanoparticles with antibody,

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transmembrane peptide or protein. However, there are few studies on the targeting of inclusion complex, which mainly through modified CD with folic acid by functional

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chemical bonds like disulfide bond to delivery drugs or DNA. Our previous study

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demonstrated that DTX and folic acid decorated CD prepared inclusion complexes, which exhibit targeting effects to cancer cells with high expression of folic acid receptors on the cell surface, such as KB cells(Xu et al., 2016b). To further

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demonstrate its targeting effect in vivo, we found that DTX/FA-CD was the most abundant and showed early accumulation, which Cy5.5/FA-CD were 1h and

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Cy5.5/CD at lease 5h, in tumor tissues in KB tumor-bearing mice by in vivo imaging system, which was significantly more than free DTX and DTX/CD group (Figure 6). We also found that the drug began re-distribution to the peri-tumorous tissues after treatment with DTX/CD at 12 h. These may be caused by enhanced permeability and retention began to weaken, and the drug in tumor tissue was saturated. These observations suggested that DTX/FA-CD not only has passive targets but also achieves excellent FA-target effects(Mei et al., 2015). Apoptosis is the major cell death pathway and occurs through a high regulated series of gene activation and expression pathways. Previous studies also showed that 11

ACCEPTED MANUSCRIPT FA-CD/DTX acts mainly through the apoptotic pathway to kill KB cells(Xu et al., 2016b). The mitochondrial pathway inducing apoptosis of cells were as importance pathways of cell apoptosis. Mitochondrial-mediated apoptosis often leads to inhibition of the mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factors. Increased ROS production, the anti-apoptotic Bcl-2

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proteins and pro-apoptotic Bax family proteins can influence the mitochondrial permeability transition pore, which opens and reduces the mitochondrial

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transmembrane potential and increases cytochrome c release. Cytochrome c can bind Apaf1 or pro-caspase 9, further activating caspase 3 and triggering the caspase

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cascade reaction, and eventually leading to apoptosis(Bhola and Letai, 2016; Giampazolias and Tait, 2015). In the present study, treatment with DTX/FA-CD

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caused a significant increase in ROS production (0.01
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decrease in ΔΨm (0.001
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cells after treatment with DTX/FA-CD（Figure 3）. Consistent with our results, Bcl-2 proteins was decrease and pro-apoptotic proteins, such as Bax, p53 and caspase 3/8， were increase after KB cell treated with DTX/FA-CD (Figure 5). Those results

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suggest that the DTX/FA-CD were treated KB cells with different concentration and time, which via mitochondrial pathway induce cell apoptosis, and express time- and

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concentration-dependent. After DTX/FA-DTX treated, the protein of p53 was increased, and it can lead to glutathione peroxidase decreased, further to reduce GSH express in KB cells. What’s more, the expression of ROS was increase follow GSH decrease, which induces the open of permeability transition pore of mitochondrial and ΔΨm decrease. Finally, the Bax protein was transferred to mitochondrial and release cytochrome C from mitochondrial to cytoplasm, result to activate signal of caspase and KB cells apoptosis. Previous reports demonstrated that DTX increased ROS production in HL-60 cells and in turn activated the caspase 3 pathway(Cao et al., 2004), similar to our results. 12

ACCEPTED MANUSCRIPT The reasons underlying the success of this DTX delivery system in achieving antitumor functions in vivo are largely attributions to FA. In the present study, we administrated different types of inclusion complexes to KB models. Based on the results in tumor volume, body weight and survival rate, the DTX/FA-CD complexes show an excellent anti-tumor effect compared with other control groups. These results

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illustrate that CD modified with FA successfully targets tumor tissue, enhances the

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accumulation time in tumors and achieves better therapeutic effect.

5. Conclusion

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In summary, our results showed that the CD inclusion complex successfully improved the chemotherapeutic potency of DTX. The CD had a folic acid decorated which was

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in favor of drug delivery to tumor. Furthermore, we determined that DTX/FA-CD kills cancer cells mainly by mitochondrial-mediated induction of cell apoptosis.

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Significant enhance DTX accumulation in tumor after FA modified CD to provide enough drug concentration and treatment time for effective cancer therapy and reduce

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side effect. Overall, these results suggested that DTX/FA-CD inclusion complex as a

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safe and efficient systemic DTX delivery system for antitumor therapy. For the next

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steps, we will deeply study the mechanism of inclusion complexes in vivo.

Conflict of interest

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The authors declare no conflict of interest.

Acknowledgements This work was supported by Science and Technology Innovation Team Project of Ningbo Science and Technology Bureau, China (No. 2015C110027), Key Laboratory of Ningbo, China (No. 2016A22002), and Natural Science Foundation of Zhejiang province, China (LQ15H300001). References

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ACCEPTED MANUSCRIPT 2016. Systematic evaluation multifunctional paclitaxel-loaded polymer-ic mixed micelles as a potential anticancer remedy to overcome multidrug resistance. Acta Biomater. S1742-7061, 30689-30684.

Figure Legends Scheme 1. Schematic diagram of DTX/FA-CD inducing KB cell apoptosis via the

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intrinsic mitochondrial pathway Fig. 1. Flow cytometry analysis of ROS production. (A) Taxotere, DTX/CD and

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DTX/FA-CD incubated for 12 h to cells with 24 μM DTX. (B) Cells were incubated with 24 μM DTX/FA-CD for various time points. (C) Cells were incubated with

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different concentrations of DTX/FA-CD for 12 h. ***p<0.0001, **p<0.01, and

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Fig. 2. Flow cytometry analysis of ΔΨm. (A) Taxotere, DTX/CD and DTX/FA-CD

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Fig. 3. GSH assays. (A) Taxotere, DTX/CD and DTX/FA-CD incubated for 12 h to cells with 24 μM DTX. (B) Cells were treated with 24 μM DTX/FA-CD for various

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times. (C) Cells were treated with different concentrations of DTX/FA-CD for 12 h. ***p<0.0001 and **p<0.01.

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Fig. 4. Catalase assays. (A) Taxotere, DTX/CD and DTX/FA-CD incubated for 12 h to cells with 24 μM DTX. (B) Cells were treated with 24 μM DTX/FA-CD for various times. (C) Cells were treated with different concentrations of DTX/FA-CD for 12 h. ***p<0.0001, **p<0.01, and *p<0.05. Fig. 5. DTX/FA-CD regulated the apoptosis proteins expression which related mitochondria-mediated apoptosis. (A) Taxotere, DTX/CD and DTX/FA-CD incubated for 24 h to cells with 10 μM DTX. (B) Cells were treated with 10 μM DTX/FA-CD for different times. (C) Cells were treated with different concentrations 16

ACCEPTED MANUSCRIPT of DTX/FA-CD for 24 h. ***p<0.0001, **p<0.01, and *p<0.05. Fig. 6. Distribution profile of Cy5.5/FA-CD in KB-bearing tumor mice after tail vein administration at a dose of 12 mg/kg. (A) Whole body fluorescence images of xenograft-bearing mice at 1, 5, 7, 12, and 24 h after intravenous injection of Cy5.5/FA-CD compared with Cy5.5/CD and free Cy5.5. (B) Fluorescence images of

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excised tumors and organs at 48 h post-injection of Cy 5.5/CD, Cy5.5 and Cy5.5/FA-CD. (C) Fluorescence images of excised tumors and organs at 1, 12, 24,

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Fig. 7. The in vivo anti-tumor therapy. KB tumor-bearing mice were subjected to

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intravenous injection of various treatments (12 mg/kg) five times at six day intervals. (A, B) Tumor volume change over the treatment period. (C) Measurement of weight

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over weekly intervals. (D) The percentages of tumor weight. (E) The tumor inhibition rate of DTX/FA-CD compared with DTX/CD and Taxotere. (F) Survival rates of treatment groups. (G) H&E analyses of tumor tissues in treatment groups. (H)

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Immunohistochemical analysis of Ki-67 in tumor tissues in treatment groups. Top

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