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Introduction

Gastric cancer (GC) is the most lethal malignancy in the digestive system (1,2). Due to the asymptomatic nature, non-specific clinical manifestations, and the lack of efficient screening programs, most GC patients were diagnosised with advanced stages. The prognosis of patients with advanced GC (AGC) is still poor even after multidisciplinary therapy combined with surgery, chemotherapy, and radiation therapy (3). Human epidermal growth factor receptor 2 (HER2) is a member of a family of receptors acting as a proto-oncogene. HER2 is overexpressed in ~20% of GC, and now it is an important target for GC. Trastuzumab, a monoclonal antibody that targets HER2, inhibits HER2-mediated signaling and thus induces antibody-dependent cellular cytotoxicity against tumor cells (4,5). The introduction of trastuzumab made a new term in HER2-positive GC.

Trastuzumab combined with chemotherapy demonstrates a significant survival benefit in patients with HER2-positive AGC in the clinic. In ToGA study (trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-esophageal junction cancer), an open-label, international, phase III, randomized controlled trial, patients with HER2-postive GC were randomly assigned to receive a standard chemotherapy regimen or chemotherapy in combination with trastuzumab. Results showed that those who received trastuzumab plus chemotherapy had prolonged overall survival (OS) (6). Furthermore, with the development of antibody-drug conjugate (ADC) technique, strategies which take advantage of selective delivery of anticancer drugs by monoclonal antibodies to antigen-expressing tumor cells have proved efficacy. For example, ado-trastuzumab emtansine (T-DM1) consists of trastuzumab linked to cytotoxic agent of emtansine as a single agent, which shows statistically significant antitumor efficacy and minor systemic toxicity in HER2-positive tumors (7–9). In GC, T-DM1 showed a promising antitumor efficacy in HER2-positive GC cell lines in vitro and xenograft tumors in vivo (10,11). Nowadays, several clinical trials are underway in patients with HER2-positive AGC, including the efficacy and safety of T-DM1 compared with paclitaxel (PTX) (9). However, T-DM1 does not show survival benefit in HER2-positive AGC as the expected remarkable efficacy for HER2-positive breast cancer (12). Therefore, HER2-directed therapies for HER2-positive GC remains a challenge.

Fortunately, nanomedicine has a significant impact on cancer therapy with the introduction of drug delivery system based on nanobiotechnology, such as nanoparticle albumin-bound paclitaxel (nab-paclitaxel). Nab-paclitaxel is a solvent-free suspension of paclitaxel and human serum albumin (HSA) with an average mini size, which allows better delivery of paclitaxel into tumors via the unique mechanism (gp60-caveolin-1-SPARC) and passive targeting of EPR (enhanced permeability and retention) effect. To date, many studies have proved the superiority of nab-paclitaxel in tumors, including GC (13–18). However, its clinical success is stilled limited by unfavorable pharmacokinetics, suboptimal biodistribution and toxicities (19). Antibody-nanoparticle conjugate (ANC) offers opportunities for optimized targeted cancer treatment (20,21). In view of these studies, here, the conjugation of the two clinical drugs of nab-paclitaxel and trastuzumab is presented as an ANC single-agent (trastuzumab/nab-paclitaxel) for precisly targeted therapy of HER2-positive GC.

Materials and methods

Main materials

Abraxane® [paclitaxel for injection (albumin bound), lot no. 6109342] was from Celgene (Fresenius Kabi USA, LLC Melrose Park, IL, USA). Taxol® (paclitaxel injection) was from Bristol-Myers Sguibb (Corden Phama Latina S.P.A, Sermoneta, Italy). Herceptin® (trastuzumab, lot no. ab2428) was purchased from Abcam. FBS (fetal bovine serum, lot no. 1698221) was purchased from Gibco Chemical Co. (Carlsbad, CA, USA). Pen Strep (penicillin streptomycin, lot no. 1665735), 0.25% trypsin-EDTA and PBS (phosphate-buffered saline, lot no. AAL211089) were purchased from HyClone (GE Healthcare Life Sciences). Matrigel basement membrane matrix (lot no. 356324) was from Corning Inc. (Corning, New York, NY, USA). EDC (C8H17N3HCL, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, cat. no. 25952-53-8) and NHS (C4H5NO2, N-hydroxysuccinimide, cat. no. 6066-82-6) were from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories, (Kumamoto, Japan). Annexin V-FITC/PI Apoptosis Detection kit was from Nanjing KeyGen Biotech Co. (Nanjing, China). Hematoxylin-eosin staining kit from Beyotime Institute of Biotechnology (Shanghai, China). BALB/c nude mice (weighing 20-22 g, 5 weeks old, half were male and half female) were obtained from Comparative Medicine Centre, Yangzhou University (Yangzhou, Jiangsu, China). Monoclonal antibodies of Bax (cat. no. ssc-493), Bcl-2 (cat. no. sc-509), caspase-3 (cat. no. ssc-271759), caspase-8 (cat. no. sc-56071), caspase-9 (cat. no. sc-17784) and survivin (cat. no. sc-101433) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). NIR797-isothiocyanate (MW 880.14 Da) was purchased from Sigma-Aldrich Co. The transmission electron microscopic (TEM) images were obtained by a JEM-2100, JEOL Ltd. (Tokyo, Japan). Flow cytometry analysis was performed by BD FACSCalibur (BD Biosciences, San Jose, CA, USA). Cells were imaged using a confocal microscope (Zeiss LSM 710; Carl Zeiss, Oberkochen, Germany), and the tumor-bearing nude mice were imaged with a Caliper IVIS (in vivo imaging system; Perkin-Elmer, Waltham, MA, USA). All the experiments were conducted according to the manufacturer's protocols. The reagents were of analytical grade.

Preparation of ANC of trastuzumab/nab-paclitaxel

ANC of trastuzumab/nab-paclitaxel was synthesized using EDC/NHS by surface activation method. Briefly, 500 µl of nab-paclitaxel was dissolved in 1 ml of PBS followed by the addition of 100 µl NHS (5.75×10−7 g/ml) and 100 µl EDC (2.3×10−7 g/ml). After spining at 10 rpm for 120 min, 20 µl of trastuzumab (0.2 mg/ml) was added in the suspension. After further rotation at 10 rpm for 120 min at 4°C, it was ultracentrifuged at 1×104 rpm, 4°C for 15 min to remove excess EDC/NHS and unconjugated trastuzumab. The process was repeated 3 times after sonication. Further, the ANC of trastuzumab/nab-paclitaxel were resuspended in 1 ml PBS and stored at −20°C. Morphological characteristics of the ANC of trastuzumab/nab-paclitaxel were examined using a TEM. Dynamic light scattering (DLS) was performed to determine the hydrodynamic radius (Rh) of trastuzumab/nab-paclitaxel at 25°C using a Dynapro™ plate reader (Wyatt Technology, Santa Barbara, CA, USA).

Cell culture

The human HER2-postive GC cell line, NCI-N87, purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and were cultured in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum (FBS), 1% Pen Strep in a humidified incubator in 5% CO2 at 37°C.

Cell viability assay

NCI-N87 cells were seeded in 96-well plates at a density of ~5,000 cells per well and treated with different treatments, paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel, respectively. Cell without treatment were used as a control. After further incubation for 48 h at 37°C, the relative cell viability was assessed using CCK-8 assays. Briefly, after the different treatments, CCK-8 (10 µl) was added to each well and incubated for a further 2 h. Optical density (OD) at 450 nm was read on an ELx800 microplate reader (BioTek, Vermont, WI, USA), and then the cell viability was calculated as follows:

OD(450 nm in test cells)/OD(450 nm in control cells) × 100%

Cell cycle analysis

NCI-N87 cells were seeded in 6-well plates. After different treatments, cells were collected, fixed in 75% cold ethanol at 4°C for at least 2 h, washed by cold phosphate-buffered saline, stained with PI/RNase staining buffer for 15 min, and then measured by flow cytometry.

Apoptosis assay

Quantification of apoptotic cells was determined using an Annexin V-fluorescein isothiocyanate (FITC)/propidum iodide (PI) detection kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions. Cells were collected after different treatments, washed twice with cold PBS and resuspended in 100 µl binding buffer, followed by staining with 5 µl of Annexin V-FITC and 10 µl of PI solution at room temperature in the dark for 15 min. Analyses were then performed using a FACSCalibur™ flow cytometer.

Morphological characteristics of the nucleus by DAPI stain

The cells were first cultured on slides in 24-well plates at a density of 1×104 cells/well. After treatment with different agents, the cells on the slides were fixed by incubation in 4% paraformaldehyde (PFA) for 30 min. After washing with PBS three times, the cells were incubated in 1 mg/ml DAPI in methanol for 30 min in the dark. The cells were then observed with a fluorescence microscope.

Western blot analysis

After different treatments, cells were lysed, and the isolated protein was quantified using the Bradford method, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred on to a polyvinylidene fluoride membrane. After being blocked, the membrane was incubated with primary polyclonal antibodies of anti-Bax, Bcl-2, caspase-3, caspase-8, caspase-9, survivin and GAPDH overnight at 4°C, and subsequently incubated with horseradish peroxidase-conjugated IgG antibody as the secondary antibody for 1 h at room temperature. The protein bands were detected using an enhanced electrochemiluminescence detection system (ECL system; Amersham Pharmacia Biotech, Amersham, UK). After normalization using the corresponding GAPDH expression, the expression levels of Bax, Bcl-2, caspase-3, caspase-8, caspase-9 and survivin were determined using densitometry scans.

Gastric cancer xenograft model in nude mice

In vivo antitumor efficacy of paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel were evaluated through tumor-bearing mice. BALB/c nude mice were kept in filter-topped cages with standard rodent chow, water available ad libitum, and a 12-h light/dark cycle. The experiment protocol was approved by the Committee on Ethical Animal Experiments at Southeast University. The mice were fed with sterile food in a specific pathogen-free facility. All mice were injected subcutaneously with 5×106 NCI-N87 cells. The length (a) and width (b) of the tumor were measured every other day. Tumor volumes (V/mm3) were calculated using the formula: V = ½ × a × b2. When tumor volumes reached ~60 mm3 (22), the mice were randomly divided into four groups: PBS as control group, paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel (all the groups were treated with equivalent paclitaxel concentration at 20 mg/kg). The intravenous treatment was done twice a week for four times. The RTV (relative tumor volume) = VX/V1, where VX and V1 represent the volumes on day X and the first day of tumor treatment. The antitumor efficacy of tumor inhibition rate is defined as both of the tumor volume and weight inhibitory rate, which is calculated using the formula: volume inhibitory rate (%) = (1-RTVaverage experimental group/RTVaverage control group) × 100%.

In vivo imaging of NIR-797-labeled trastuzumab/nab-paclitaxel

Firstly, NIR-797-labeld trastuzumab/nab-paclitaxel were synthesised by physical adsorption. Briefly, 1 mg of NIR-797 was added to 1 ml of paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel (5 mg/ml paclitaxel equivalent) respectively. After spining at 10 rpm overnight, the suspensions of trastuzumab/nab-paclitaxel were ultracentrifuged at 10,000 rpm, 4°C for 15 min to remove unadsorpted dye, finally the precipitations were resuspended in 1 ml of PBS for injection. For in vivo imaging, the tumor-bearing mice were injected via tail vein at a single dose of NIR-797-labled paclitaxel, NIR-797-labled nab-paclitaxel and NIR-797-labled trastuzumab/nab-paclitaxel at 20 mg/kg paclitaxel equivalent concentration when the tumors reached ~60 mm3. The mice were imaged in a small animal imaging system by X-ray and fluorescence at 0, 2, 4, 8, 24, 48 and 72 h after injection.

Histopathological examination

After different treatments, the mice were sacrificed by cervical dislocation. The organs, including lung, heart, liver, spleen, kidney and tumor tissues isolated from each group were immersed in 4% PFA solution, embedded in paraffin, and stained with hematoxylin-eosin. Thereafter, the tissues were examined using an Olympus IX51 microscope (×200; Olympus Corp., Tokyo, Japan).

Statistical analysis

GraphPad Prism 5.0 (GraphPad software; San Diego, CA, USA) was used for all statistical analysis. The mean ± SD was determined for each group in the individual experiments. The Student's t-test was used to determine the significance of differences between different groups. P-values <0.05 were considered to indicate statistically significant difference.

Results

Characterization of trastuzumab/nab-paclitaxel

The scheme for the preparation of trastuzumab/nab-paclitaxel is presented in Fig. 1A. The TEM images in Fig. 1B show the trastuzumab/nab-paclitaxel is spherical in shape and in a suitable size (139.18±32.06 nm) measured by DLS (Fig. 1C) for drug delivery. The concentration of paclitaxel of trastuzumab/nab-paclitaxel by UV spectrophotometry indicates that the concentration of paclitaxel was one-tenth of nab-paclitaxel.

Figure 1. Characterization of trastuzumab/nab-paclitaxel. (A) The scheme for the preparation of trastuzumab/nab-paclitaxel. (B) TEM image. (C) Size distribution.

In vitro antitumor efficacy

To determine the antitumor efficacy in vitro, we measured the cytotoxicity following treatment of HER2-postive GC NCI-N87 cells with paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel for 48 h. As shown in Fig. 2A, typical characteristic apoptotic changes, such as chromatin condensation, convoluted nuclei with cavitations, fragmentation of the nucleus, and apoptotic bodies, could be found after different treatments. That is to say, paclitaxel, nab-paclitaxel, and trastuzumab/nab-paclitaxel could inhibit the growth of NCI-N87 cells, whereas trastuzumab/nab-paclitaxel was found to be more cytotoxic than paclitaxel and nab-paclitaxel (Fig. 2B). The half-maximal inhibitory concentration (IC50), defined as the concentration of paclitaxel to kill 50% of cells, was found to be 0.24±0.08, 0.13±0.03 and 0.048±0.01 µg/ml for paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel, respectively, with an excellent dose-effect relationship, suggesting that the killing effects of these drugs were dose-dependent. Similarly, the apoptosis rate was higher in trastuzumab/nab-paclitaxel group (51.30±2.28%) than paclitaxel group (43.32±1.08%) and nab-paclitaxel group (46.64±1.47%) in NCI-N87 cells (Fig. 2C). These data clearly suggest that trastuzumab/nab-paclitaxel could enhance cytotoxic effect against NCI-N87 cells better than paclitaxel and nab-paclitaxel in vitro.

Figure 2. Antitumor efficacy of trastuzumab/nab-paclitaxel in vitro. (A) Morphology of nuclear of NCI-N87 cells treated by paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel. PBS as control (bar, 20 µm). The white arrows indicate chromatin fragmentation of nucleus. (B) Cytotoxic effect of paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel. (C) Apoptosis rate of NCI-N87 cells with DAPI staining by paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel. PBS as control.

Cell cycle distributions of NCI-N87 cells

We quantified the population of NCI-N87 cells in different stages of the cell cycle upon treatments with paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel, respectively, at a concentration of 0.24 µg/ml paclitaxel equivalent for 48 h. In our study, after different treatments, G2/M arrest was frequently observed in NCI-N87 cells. Moreover, trastuzumab/nab-paclitaxel showed more significant G2/M arrest (56.35±2.14%) than that of paclitaxel (18.17±1.34%) and nab-paclitaxel (44.83±2.58%), which is shown in Fig. 3.

Figure 3. Cell cycle distributions of NCI-N87 cells by different treatments. (A) Cells were exposed to different treatment (PBS, paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel) for 48 h, followed by analysis of cell cycle by flow cytometry. (B) Distribution of NCI-N87 cells at different phases of the cell cycle distribution with different treatment (PBS, paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel).

Apoptosis induction in NCI-N87 cells

Similar to the two clinical drugs paclitaxel and nab-paclitaxel, the novel ANC of trastuzumab/nab-paclitaxel could also induce apoptosis in NCI-N87 cells (Fig. 4A). The early apoptotic rates of NCI-N87 cells treated by trastuzumab/nab-paclitaxel (20.8±0.28%) is higher than that of nab-paclitaxel (14.9±0.17%), paclitaxel (12.7±0.65%), and PBS as control (2.8±0.12%) (Fig. 4B). To explore the possible signaling pathways involved in apoptosis, we examined the changes of the apoptosis-related proteins, including caspase-3, caspase-8, caspase-9, Bax, Bcl-2, and survivin by western blotting. As shown in Fig. 4C and D, when treated with paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel for 48 h, the levels of both Bcl-2 and survivin in NCI-N87 cells were significantly downregulated, while those of Bax, caspase-3, caspase-8, caspase-9 protein were upregulated as well as the ratio of Bax/Bcl-2.

Figure 4. Induction of apoptosis in NCI-N87 cells by different treatments. (A) NCI-N87 cells were treated with paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel at concentration of 0.24 µg/ml for 48 h, while PBS group was the control. (B) The apoptosis rates are calculated by adding up the percentage of early apoptosis rate and later apoptosis rate. (C) Western blot analysis of endogenous and different treatments (a) PBS, (b) paclitaxel, (c) nab-paclitaxel, (d) trastuzumab/nab-paclitaxel)-induced Bax, Bcl-2, caspase-3, caspase-8, caspase-9 and survivin protein levels in NCI-N87 cells. (D) The relative intensity of Bax, Bcl-2, caspase-3, caspase-8, caspase-9 and survivin protein after 48-h treatment.

In vivo antitumor efficacy

Antitumor efficacy of trastuzumab/nab-paclitaxel compared with paclitaxel and nab-paclitaxel were further evaluated in NCI-N87 xenograft models at a 20 mg/kg paclitaxel equivalent dose in vivo. During the experiment period, all the mice were weighed and tumor volumes were measured every other day. At 4 weeks after treatment, mice were sacrificed. Then the mouse tumors were imaged (Fig. 5A) and tumor weight was recorded (Fig. 5B). Mean tumor volume treated with trastuzumab/nab-paclitaxel was 233±24 mm3, nab-paclitaxel of 559±97 mm3, paclitaxel of 871±94 mm3 and PBS as control of 1,576±190 mm3 (Fig. 5C). Obvious significant tumor regression was obtained in mice treated with trastuzumab/nab-paclitaxel compared with paclitaxel and nab-paclitaxel. In addition, trastuzumab/nab-paclitaxel compared with nab-paclitaxel and paclitaxel did not have significanly increased body weight (Fig. 5D).

Figure 5. The antitumor effect of different treatments on NCI-N87 human xenograft models. (A) At the experimental end-point, tumors were dissected and photographed (n=3 per group). Only one mouse is demonstrated as an example for each group. (B) The tumor weight were compared with Student's t-test (n=3, *P<0.05). (C) The mice transplanted with NCI-N87 xenograft were randomly divided into two groups and given injection of trastuzumab/nab-paclitaxel compared with paclitaxel, nab-paclitaxel and PBS (20 mg/kg per week for a period of 4 weeks). The tumor volumes were expressed as mean ± SD (n=3, *P<0.05, **P<0.01). (D) The average body weight of each group was expressed as mean ± SD (n=3).

NIRF imaging and biodistribution of NIR-797-labeled drugs in tumor-bearing mice

To visualize the biodistribution of trastuzumab/nab-paclitaxel in NCI-N87 tumor-bearing mice, a near-infrared fluorescent (NIRF) dye, NIR-797, was labeled to paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel. NIR fluorescence signals were clearly and dynamically observed in the mice. As shown in Fig. 6, 2 h after injection, these fluorescence signals of NIR-797-labeled paclitaxel were strong and mainly localized in the body area, but it began to decrease after 8 h and decreased gradually especially in the tumor site with no distribution in the brain. Obvious increased accumulation of NIR fluorescence signals was observed in the tumor site of the mice injected nab-paclitaxel and trastuzumab/nab-paclitaxel. What is inspiring, as time increased, the fluorescence signals of tumors became stronger and the fluorescence signals of liver began to weaken. Fluorescence signals of NIR-797-labeled trastuzumab/nab-paclitaxel remains strong until 48 h after injection, at which point the fluorescence signals of NIR-797-labeled nab-paclitaxel has already decreased. In addition, 72 h after injection, fluorescence signals could only be seen in the liver and tumor site which indicated a better targeting and sustained release of trastuzumab/nab-paclitaxel. Furthermore, these fluorescence signals of trastuzumab/nab-paclitaxel group were only observed in liver and tumor areas.

Figure 6. NIRF images of NCI-N87 tumor-bearing mice after treatments. (A) NIR797-labeled paclitaxel. (B) NIR797-labeled nab-paclitaxel. (C) NIR797-labeled trastuzumab/nab-paclitaxel. The color depth in tumor tissue indicates the drug concentration in that area.

Histopathological examination

We carried out histological bioanalysis of organs to evaluate the potential side effects of trastuzumab/nab-paclitaxel on the main organs of mice in vivo. As shown in Fig. 7, there were no apparent histopathologic changes in the tissues, including heart, liver, spleen, lung and kidney.

Figure 7. Histopathological examination of organs and tumor tissues in NCI-N87 xenograft model after the treatment (hematoxylin-eosin staining, ×200). (A) PBS. (B) Paclitaxel. (C) Nab-paclitaxel. (D) Trastuzumab/nab-paclitaxel.

Discussion

AGC remains one of the most lethal malignancies due to its intrinsic resistance and its aggressiveness to standard chemotherapy and targeted therapy. Nab-paclitaxel as carriers of chemotherapeutic drugs to reverse the toxicity of paclitaxel have displayed that nab-paclitaxel can significantly increase the antitumor effection and still maintain a certain concentration. In clinic, trastuzumab combined with chemotherapy as ADC demonstrates a significant survival benefit in patients with HER2-positive AGC. Thus, in this study, we further constructed the ANC of trastuzumab/nab-paclitaxel for AGC.

Apoptosis is the preferred anticancer manner regulated by genes (23–25). Many oncogenes and tumor suppressor genes are involved in the regulation of apoptosis; the proto-oncogene Bcl-2 is the most important (26). Bcl-2 and Bax are a pair of positive and negative regulators of genes; Bax induced apoptosis, while Bcl-2 inhibited apoptosis (27). Caspase-3 may promote apoptosis, while survivin is the strongest inhibitor of apoptosis in AGC, also promoting cell proliferation, which can directly inhibit the activity of caspase-3 and caspase-8 and caspase-9, thereby blocking the apoptotic process (28,29). These results of apoptosis phenomenon in NCI-N87 cells treated with PBS, paclitaxel, nab-paclitaxel and trastuzumab/nab-paclitaxel by western blotting observation showed that ANC of trastuzumab/nab-paclitaxel could decrease Bcl-2 and survivin protein expression, and increase Bax and caspase-3 protein expression; the difference was more significant than the control group. Caspase activation is generally considered to be the hallmark of apoptosis, and caspase-3 is the main effector caspase that is involved in apoptosis. These results indicate that trastuzumab/nab-paclitaxel could induce anticancer activity in a caspase-dependent manner, and effectiveness of the survivin pathway.

Paclitaxel's mode of antitumor action is the disruption of microtubule dynamics, and paclitaxel is believed to mediate G2/M cell cycle arrest in various cancer cells, including GC (30–32). In our study, after different treatments, G2/M arrest was frequently observed in NCI-N87 cells. Furthermore, trastuzumab/nab-paclitaxel showed more significant G2/M arrest (56.35±2.14%) than that of paclitaxel (18.17±1.34%) and nab-paclitaxel (44.83±2.58%). Conjugation of paclitaxel and trastuzumab as an ADC was thought to be promising and worthy (33,34). However, clinical results from ADC failed to demonstrate therapeutic benefit with shortcomings of poor in vitro potency, modest in vivo activity and localization in human tumors (35). Albumin-bound formulation for paclitaxel based on nano-drug delivery system, nab-paclitaxel, has the advantage of passive target for tumor due to EPR effect. Now, deliberate modifications of ligand or antibody to the surface of nanoparticles were conducted to achieve specific targeting to the corresponding receptor on tumor cells (22,36). The unique targeting mechanism of HSA (22,37) and passive targeting property of nab-paclitaxel along with the active targeting property of anti-HER2 antibody may introduce sequentially dual-targeting and more precise paclitaxel delivery.

Currently, the NIRF imaging technique is widely used for cancer molecular imaging research; it has a clearer and longer visualization for tracking in vivo. In our study, fluorescence signals of NIR-797-labeled trastuzumab/nab-paclitaxel remains strong until 48 h after injection. Furthermore, the fluorescence signals of trastuzumab/nab-paclitaxel group were only observed in liver and tumor area, illustrating that the ANC of trastuzumab/nab-paclitaxel could target the tumor tissue more precisely than nanoparticles. In vivo antitumor efficacy demonstrated that the ANC of the trastuzumab/nab-paclitaxel had more significant tumor regression than other treatment groups. In addition, trastuzumab/nab-paclitaxel compared with nab-paclitaxel and paclitaxel had no obviously effect on the quality of life.

In conclusion, this study successfully synthesized antibody-nanoparticle conjugate of trastuzumab/nab-paclitaxel with properties of passive and active target. In vitro and in vivo findings illustrated that trastuzumab/nab-paclitaxel could enhance antitumor efficacy, which could represent a novel precisely targeting therapeutic agent for HER2-positive GC.

Acknowledgements

This study was supported by the National Nature Science Foundation of People's Republic of China (81371678).

Glossary

Abbreviations

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References