All reagents and solvents were used as received, without further purification. Monomethoxy PEG with a molecular weight of 20,000 kDa (mPEG 20,000), D,L-lactide and stannous octoate were purchased from Sigma-Aldrich Chemical Corp. (Shanghai, China); DTX was purchased from Beijing Norzer Pharmaceutical Co., Ltd. (Beijing, China); free-DTX (Aisu^®) is manufactured by Hengrui Pharmaceutical Co., Ltd. (Jiangsu, China); and 3-(4,5)-dimethylthiazol(z-y1)-3,5-di-phenyltetrazolium bromide (MTT) was obtained from Amresco (Solon, OH, USA); Annexin V-FITC apoptosis detection kit was purchased from 4A Biotech Co., Ltd. (Beijing, China); VivoGlo^® luciferin was purchased from Promega Corporation (Madison, WI, USA). Trypsin, fetal bovine serum (FBS) and RPMI-1640 medium were purchased from HyClone (Logan, UT, USA), and culture flasks and dishes were from Corning (Corning, NY, USA).

Human non-small lung cancer A549 cell line was provided by the Department of Pathology in Institute of Medicinal Biotechnology in Peking Union Medical College. A549 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. Female BALB/c mice (6–8-weeks old) were used for antitumor efficacy studies and were purchased from Beijing Vital River Laboratories (Beijing, China).

Animals were acclimatized in the holding facility prior to beginning of the present study. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences. All surgeries were performed under sodium pentobarbital anesthesia (5 mg/ml solution), and all efforts were made to minimize suffering. Lung tumor and other sections were routinely stained with hematoxylin and eosin (H&E) and evaluated under a light microscope.

DTX was dissolved in chloroform and ethanol to form solution A, albumin was dissolved in sterile water to form solution B. The solution A and B was mixed and stirred by homogenate machine 5 min to form raw milk, the raw milk was moved into high pressure homogenizer, under the 20,000 psi for 12 cycles. The chloroform of mixture was eliminated using rotary evaporator for 25 min and followed by filtration through a 0.22 µm filter.

DANPs and mPEG (20,000 kDa) were added to the solution of boric acid buffer (0.1 mol/l, pH 9.0) according to the ratio of 3:1 with stirring and the reaction was terminated via adding glycine (1 mg/ml) 3 h later. Unbound HAS and PEG were removed by ultrafiltration (MWCO, 70 kDa) (21,22).

Determination via PAGE gel electrophoresis: DANPs and PEG-DANPs were determined via PAGE gel electrophoresis with iodine staining and Mas blue staining.

The in vitro cytotoxic activity of DANPs and PEG-DANPs was evaluated by the MTT assay. Briefly, the A549 cells (8×10⁴ cells/ml) were seeded into 96-well plates and incubated for 24 h to allow cell attachment. The cells were then treated with a series of phosphate-buffered saline (PBS) (control), Aisu^®, DANPs and PEG-DANPs at 37°C (0.001, 0.01, 0.1, 1, 10 and 100 µg/ml). At the incubation-time points of 48 h, 20 µl of MTT (5 mg/ml) was added and incubated for 4 h, MTT was aspirated off and 180 µl/well of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, and the plate was gently shaken for 10 min. The optical density (OD) was measured at 490 nm by Synergy H1m monochromator-based multi-mode microplate reader (BioTek, Winooski, VT, USA). The cell inhibition was calculated according to the below formula: Cell inhibition (%) = [1 − (OD_sample − OD_blank)/(OD_sample − OD_blank)] × 100%. The results were expressed as means ± SD of 3 measurements. No precipitation of DTX was found during incubation procedure (23).

Apoptotic cells were determined by dual staining with an Annexin V and propidium iodide (PI) kit (4A Biotech Co., Ltd.) according to the manufacturer's instructions (24). After 48 h of incubation in the exponential stage, A549 cells seeded in 12-well plates were treated for a further 48 h with 10 nmol/ml Aisu^®, DANPs and PEG-DANPs, respectively. After treatment, cells were washed twice with warm PBS, detached by trypsin without EDTA, then through the following steps: collection, centrifugation, washing with warm PBS, further staining with PI and Annexin V-FITC for 15 min at room temperature in the dark. Apoptosis was then analyzed using a FACScan cytometer. Quadrant analysis was performed and cells that stained positive for both Annexin V-FITC and PI were designated as apoptotic, while unstained cells were designated as viable.

All experimental procedures were performed in conformity with institutional guidelines and protocols for the care and use of laboratory animals. We chose 40 BALB/c mice to divide into two parts, one part was to establish the transplantation tumor model (25,26) to observe the inhibition of drugs for transplantation tumor and weight changes of nude mice; the other part was to establish the in situ carcinoma model to observe the inhibition of drugs for in situ carcinoma and metastasis. Each group contained 20 mice and they were equally divided into four groups, respectively, negative control (glucose injection group), positive control (Aisu^® group), DANPs and PEG-DANPs groups with 5 animals each. The lung cancer A549 cells were suspended in BD Matrigel, and the mice in each group were implanted with 3×10⁶ cells in alar or in the lung. When the tumor volume in transplantation tumor group reached ~120 mm³, the mice were treated 4 times at 7-day intervals with 5% glucose injection (negative control), with Aisu^®, DANPs or PEG-DANPs at the same time in both parts, respectively. All formulations were injected intravenously via the tail vein at a DTX dose of 20 mg/kg. The body weight and tumor volume were measured simultaneously. Tumor volume was calculated using the equation of V = w² × l/2. Here, w and l are the width and length of the tumor. Forty-eight hours after the last treatment, the mice were sacrificed. The tumors with lung and other major organs (including heart, liver, spleen and kidney) were removed, fixed in 10% formalin solution, and subjected to paraffin embedding for H&E staining.

Additional 32 nude mice were used to establish the transplantation tumor model and divided into negative control group (group glucose injection), positive control group (Aisu^®), DANPs and PEG-DANPs group, the mice were treated until natural death, observe and the survival rate was drawn.

Results are presented as means ± SD. Statistical comparisons were carried out by t-test or ANOVA analysis. The level of significance was set at p<0.05.

The PAGE gel electrophoresis displayed PEG-DANPs existing in two bands, PEG and protein, respectively; whereas there was only one band in DANPs, which represents the protein (Fig. 1).

Fig. 2 shows the result of the cytotoxicity of Aisu^®, DANPs and PEG-DANPs against A549 lung cancer cells. A549 cells were exposed to a series of equivalent concentrations of Aisu^®, DANPs and PEG-DANPs for 48 h, and the inhibition rates were determined via the MTT method. The cell survival rate had a dose-dependent inverse relationship with the drug concentrations. PEG-DANPs accelerated cellular uptake of the drug and induced higher cytotoxicity in cancer cells than Aisu^® and DANPs, particularly at lower DTX concentrations (0.001–0.1 µg/ml). However, A549 cells were more sensitive to Aisu^® than DANPs and PEG-DANPs at higher concentrations (1–100 µg/ml). Nanoparticles are internalized into cancer cells via endocytic mechanisms (27), while the free-drug diffuses into cells according to the concentration gradient between the intracellular and extracellular environments. It is why Aisu^® is more cytotoxic at higher concentrations.

DTX was described as an antimitotic agent which could bind to β-tubulin, resulting in blocking the cell cycle at the G2/M phase and apoptosis of cells (28,29). According to a previous study, encapsulation of DTX in nanoparticles could increase apoptosis of prostate cancer cells (30). Given that PEG-DTX-HANPs demonstrated stronger in vitro cytotoxicity than DANPs and Aisu^®, we performed apoptosis assays using Annexin V-FITC and PI staining to compare apoptosis induction. As predicted, PEG-DANPs (55.65%) increased late apoptosis in A549 cells compared with DANPs and Aisu^® (25.85 and 43.00%) (Fig. 3).

Tumor-bearing nude mice were injected with Aisu^®, DANPs and PEG-DANPs, and their therapeutic effects were examined by measuring the suppression of body weight and tumor growth. The body weight of the mice in the negative control groups was basically unchanged or slightly increased, while the mice in the drug groups all lost weight in the process of the treatment. However, the reduction range in the Aisu^® group was more significant than the other two groups (p<0.01) (Fig. 4), suggesting severe systemic toxicity in addition to tumor toxicity. It was found that all the tumor volumes treated with Aisu^®, DANPs and PEG-DANPs were much smaller than those of negative control groups treated with the same dose (p<0.05), and the tumors of PEG-DANPs groups were obviously smaller than those of Aisu^® groups (Fig. 5A and B), indicating that PEG-DANPs is the most effective to inhibit tumor growth among the three.

A typical tumor cellular morphology was seen under the microscope in the negative control groups both in transplant model and in situ. Regardless of the tissue, tumor or lung, we could see that the tumor cells were of different sizes with abundant deep blue stained nuclei, and the nuclear division was significant. There was also loss of polarity, tightly packed cells with ill-defined nuclei and visible focal degeneration or necrosis of cancer cells. This showed that the model had been established successfully (Fig. 6A and B). In the PEG-DANPs groups, the number of cancer cells was significantly reduced, the color of nuclei was stained red rather than light blue in DANPs and Aisu^® group, and large necrotic areas could be seen without any nuclear division.

Extensive metastases were found in the livers of control group mice, and there were large tumor nests, we could also see liver metastases both in Aisu^® and DANPs-treated mice which were significant smaller than the control groups. In contrast, liver tissue from PEG-DANP-treated mice showed barely measurable levels of tumor cells but only few neutrophils infiltrated by tumor cells (Fig. 7), these results suggest that PEG-DANPs could suppress metastases in other organs such as liver more effectively than DANPs and Aisu^®.

Nude mice in control group were all dead on the 36th day, and the mice in PEG-DANPs group were dead on the 56th day. Fig. 8 shows that the mice which were treated with PEG-DANPs survived longer than the other groups, and the differences are significant.