Chitosan with a deacetylation degree (DD) of 95.3% and the molecular weight (Mw) of 6-270 kDa was purchased from Kabo Biochemical Company (Shanghai, China). Polyaspartic acid was prepared by the Department of Macromolecular Science, Key Laborotory of Molecular Engineering of Polymers of Chinese Ministry of Education, Fudan University (Shanghai) as previously described[16]. The average molecular weight of PAsp was 5.0 kDa. 5-fluorouracil was purchased from Donghai Pharmaceutical Company (Shanghai, China).The human gastric cancer cell line, SGC-7901, was serially subcultivated by the Department of Laboratory of Gastroenterology, Zhongshan Hospital. Nude male BALB/c mice (aged 35-42 d and weighing 20-23 g) and Kunming male mice (weight 5-20 g) were obtained from the Department of Laboratory Animals, Fudan University. Methyl cellulose M450 was purchased from China Medicine (group) Shanghai Chemical Reagent Corporation. Iscove’s modified Dulbecco’s medium was purchased from GIBCO Company (UK). Fetal bovine serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co, Ltd (Hangzhou, China). Antisubstance R-0023 was purchased from Changdao Biotechnology Ltd (Shanghai). All other chemicals were of analytical grade and used without further purification.

Chitosan nanoparticles were prepared as reported by Calvo et al[17] (1997) based on the ionic gelation of CS with TPP anions. Briefly, chitosan was dissolved in dilute acetic acid solution (10 g/L). The concentration of acetic acid in aqueous solution was the same as that of chitosan. Quantum satis 5-Fu was dissolved in this solution at room temperature. Afterwards, under magnetic stirring, the mixture solution of 5-Fu and CS was dropped into the Pasp solution at a rate of one drop/sec. Then, opalescent suspension was formed. The obtained suspension was filtered with a paper filter for use. Glutaraldehyde crosslinking nanoparticles were dropped in to drug-loaded CTS-Pasp suspension under magnetic stirring. This mixture was further stirred for three hours at room temperature. CTS-Pasp-5-Fu nanoparticles were separated from the aqueous suspension medium by ultra-centrifugation at 35 000 r/min for 30 min at 25°C, washed by dilute acetic acid (pH 5.0) solution and separated by three times ultracentrifugation. The sample was re-dispersed.

Dynamic light scattering (DLS) (Malvern, Autoszer 4700) was used to measure the hydrodynamic diameter and size distribution[18]. DLS measurement was done with a wave length of 532 nm at 25°C with an angle detection of 90^°.

The morphology and dried TEM-assessed size measurement of the CTS-Pasp-5Fu nanoparticles were examined under transmission electron microscope (TEM; Hitachi, H-600). The sample was dried at room temperature and examined using a TEM without staining.

The encapsulation efficiency and loading capacity of nanoparticles were determined by separation of nanoparticles from the aqueous medium containing non-associated 5-Fu by ultracentrifugation at 35 000 r/min for 30 min at 14°C. The amount of free 5-Fu in the supernatant was measured by HPLC. HPLC was performed using a LC-4A HPLC system equipped with a LC-4A pump, and a SPD-10A UV detector (Shimadzu, Kyoto, Japan). The detective wavelength was set at 270 nm. HPLC analysis of samples was performed using a Science C18 column (4.6 × 250 nm, 5 &mgr;m, Japan) preceded by a C18 guard column(GL Science, Japan).The column temperature was maintained at 30°C. The mobile phase was a mixture of methanol/3.6% acetic acid. The flow rate was 1.0 mL/min. The 5-Fu encapsulation efficiency (EE) and the 5-Fu loading capacity (LC) of the nanoparticles were calculated as follows:

EE = (The amount of 5-Fu in the nanoparticles/Total amount of 5-Fu) × 100%

LC = (The amount of 5-Fu in the nanoparticles/Total amount of nanoparticles weight) × 100%

All measurements were performed in triplicate.

In vitro 5-Fu release profiles of chitosan-Pasp-5-Fu nanoparticles were determined as follows. The CTS-Pasp-5-Fu nanoparticles were separated from the aqueous suspension medium through ultra-centrifugation. CTS-Pasp-5-Fu nanoparticles and 5-Fu were re-dispersed in 4.0 mL of phosphate buffer saline (PBS), respectively, and placed into a dialysis membrane bag with a molecular weight cut-off of 10 kDa, tied and placed into 40.0 mL PBS medium. The entire system was kept at 37°C with continuous magnetic stirring. At 15 min, 1, 4, 8, 24, 48, 96, 144 or 192 h, 3 mL release medium was removed at each time point and 3 mL fresh medium PBS solution was added into the system. The amount of 5-Fu in the release medium was evaluated by HPLC. All measurements were performed in triplicate.

Kunming male mice (weight 20-25 g) were randomly divided into two groups. Each group was administrated with 5-Fu and CTS-Pasp nanoparticles. The plasma concentrations of 5-Fu were evaluated by HPLC after 15 min, 1, 2, 4, 6, 8, 12, 16, 24 and 48 h to compare their concentration curves[9].

Nude BALB/c male mice (aged 35-42 d and weighing 20-23 g) were inoculated subcutaneously near the nape with the transplanted human SGC-7901 gastric carcinoma cell line (1 × 10⁷ cells per mouse). Two weeks later, the exuberantly proliferating tumor tissues were cut into 1.5 mm thick pieces and inoculated subcutaneously near nape of the 32 nude male BALB/c mice (aged 33-42 d and weighing 20-23 g) under aseptic conditions. The diameter of the tumor tissue transplanted into each nude mouse was measured with a slide caliper rule.

When the tumors grew to 100-300 nm³, the animals were randomly allocated into 4 groups with 8 mice in each group: CTS-Pasp-5Fu (containing 5-Fu 1.25 g/L), 5-Fu (1.25 g/L), chitosan-polyaspartic acid, and normal saline groups. Tumor weight was measured and the diameter measurement method was used to observe the dynamic changes in the antitumor response. Twenty-five mg/kg CTS-Pasp-5-Fu and 0.025 g/kg 5-Fu were given[19] each time whereas the control group was given an equal amount of normal saline or CTS-Pasp by gastric perfusion. The formula for calculating the tumor volume (TV) is: TV = 1/2 × a × b², where a and b represent the length and width[20].

The inhibition rate of tumor growth (IR)[20] was calculated based on the results of the measurements. IR was calculated using the following formula: IR = (Tc-Tt)/Tc × 100%, where Tc represents control group and Tt represents treatment group.

The tumor was weighed when the experiment was over and the tumor inhibition rates (TIR) were calculated according to the following formula: TIR = (1-tumor weight of treatment group/tumor weight of control group) × 100%.

The white blood cell (WBC) count, total bilirubin (TB), alanine aminotransferase (ALT) and creatinine in the four groups were detected after 2 wk.

The nude mice were killed under aseptic conditions. The mice were sterilized thoroughly with 70% alcohol and the pelt was peeled off to fully expose the hip joint. Sterile sharp dissecting scissors were used to cut the knee joint and the femur near hip joint. The tissues were removed and bones were placed in DMEM culture solution. The ends of the bones were trimmed, a 1 mL sterile syringe needle was inserted into marrow shaft at the end of the femur and the marrow was flushed with Iscove’s MDM with 2% FBS in a sterile tube. Flushing was repeated and the number of nucleated cells were counted. The bone marrow cell suspension was done at a concentration of 2 × 10⁵/mL and 0.3 mL of those cells was added into 3 mL MethoCult^TM medium (prepared by Department of Gastroenterology, Zhongshan Hospital, Fudan University). The tubes were thoroughly mixed and let stand for 2-3 min to allow bubbles to rise to the top. MethoCult^TM of 1.1 mL was dispensed into each 35 mm dish. Cultures were placed in a water-jacket at 37°C, 5% CO₂, ≥ 95% humidity for 7 d. The growth of bone marrow cell colonies (≥ 30 nucleated cells constituted one colony) was observed[21].

The tumors, liver and nephridial tissues of the four groups were collected, fixed with 10% formalin, stained with hematoxylin and eosin, paraffin embedded and sectioned. The structural change of cells and tissues was observed. Four-grade histological denominator was used: +++, total necrosis; ++, necrosis areas exceed half of total areas; +, necrosis areas less than half of the total areas; -, no necrosis[22].

Tumor tissues from the four groups were paraffin embedded and shaken. The sections were deparaffinaged and douched with PBS 5 minutes after quenching the endogenous peroxidate with 3% hydrogen dioxide. They were repaired twice with citric acid fluid for 15 min, added with the first antisubstance (Bcl-2, 1:100; Bax, 1:100) and stayed overnight at 4°C. The second antisubstance with HRP was incubated for 30 min at 37°C and stained with a diaminobenzidine (DAB) developer. The sections were bleached with alcohol, cleared with methyl benzene, mounted with neutral balsam and photographed (ZEISS microscope, Germany). The ratio of positive expression area to the total image area was calculated.

The SPSS software package (SAS Institute Inc, North Carolina, USA) was used for analysis of variances. P < 0.05 was considered statistically different.

The mean hydrated size of particles and distribution of CTS-Pasp-5Fu nanoparticles were determined by DLS. The results indicated that the mean size and distribution of the samples were 206 nm and 0.14, respectively. The encapsulation efficiency (EE) of CTS-Pasp-5Fu was 40.2%, and the loading capacity was 34.9%. Figure 1 shows the morphological characteristics of CTS-Pasp-5Fu nanoparticles which were synthesized by ion gelatinization. The nanoparticles were spherical in shape and well distributed. The diameter of the particles ranged from 150 to 250 nm. 5-Fu was dispersed in nanoparticles by electrostatic interaction with particulate form.

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Figure 1 TEM photography of CTS-Pasp-5Fu nanoparticles showing. It shows the morphological characteristics of CTS-Pasp-5Fu nanoparticles synthesized by ion gelatification. A: The nanoparticles are in regular spherical shape and well distributed; B: The diameter of the particles is between 150-250 nm.

Figure 2 shows the release profile of 5-Fu and CTS-Pasp-5-Fu. All the pure 5-Fu released rapidly from the dialysis bag within the first 1 h. CTS-Pasp-5Fu nanoparticles released 5-Fu gradually up to 100% in 100 h. Compared with the drug release of pure 5-Fu with 5-Fu loading CTS-Pasp nanoparticles, CTS-Pasp-5Fu nanoparticles might be used to provide a continuous release. We considered that 5-Fu embedded into the nanoparticles might be bound to PAsp by ionic reaction resulting in slow 5-Fu diffusion. 5-Fu released slowly and incompletely.

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Figure 2 Typical in vitro release profile of pure 5-Fu (▪) and CTS-Pasp-5Fu (▲) in PBS. All pure 5-Fu escaped rapidly from the dialysis bag within the first 1 h. CTS-Pasp-5Fu nanoparticles released 5-Fu gradually up to 100% in 100 h.

Figure 3 shows the concentration-time curves for the 5-Fu and CTS-Pasp-5Fu groups. The maximal concentration (Cmax) in the 5-Fu group occurred within 15 min and decreased rapidly, while that in the CTS-Pasp-5-Fu group occurred 6 h after the administration and the effective concentration lasted about 14 h. The plasma Cmax in the CTS-Pasp-5Fu group was lower than the 5-Fu group.

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Figure 3 The concentration-time curves of 5-Fu group and CTS-Pasp-5Fu nanoparticles group (48 h). The maximal concentration (Cmax) of 5-Fu group occurred within 15 min and decreased rapidly, and that of CTS-Pasp-5Fu nanoparticles group occurred 6 h after the administration and the effective concentration lasted about 14 h.

Before the treatment, the weight and tumor volume in four groups were not significantly different (P > 0.05). After the treatment, the mice in the 5-Fu group lost weight gradually (Table 1) and became hypokinetic and were in a poor general state. The other three groups were in a good general state. At the end of the experiment, 6 of 8 nude mice survived in the 5-Fu group and no mouse died in the other three groups. The inhibition rate of tumor growth (IR) for the 5-Fu and CTS-Pasp-5Fu groups was significantly higher than that for the NS group (58.69%, P = 0.004; 70.82%, P = 0.00015; Table 2). The IR for the CTS-Pasp-5Fu group was also enhanced but was not significantly different from that for the 5-Fu group (P > 0.05, P = 0.206; Table 2). There was no difference in the IR between CTS-Pasp and NS groups (P > 0.05, P = 0.607). The tumor size in the NS group was bigger than that in the 5-Fu and CTS-Psap-5FU groups (all P < 0.001, Table 3), and the tumor weight of CTS-Pasp and NS groups was not different (P > 0.05). Tumor inhibition rate (TIR) for the CTS-Pasp-5Fu and 5FU groups was significantly higher than that for the NS group (72.79% and 65.3%; Table 3). TIR for the CTS-Pasp-5Fu group was higher than that for the 5-Fu group, but the difference was not significant (Table 3, Figure 4).

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Figure 4 Tumors in four groups.

Figure 4 shows all the tumors of the four groups at the end of the experiment. There were only 6 tumors left in the 5-Fu group. The tumors of those of NS and CTS-Pasp groups were obviously bigger than the other two groups. The size of tumors in the 5-Fu and CTS-Pasp-5Fu groups was not significantly different, but TIR for the CTS-Pasp-5Fu group was higher than that for the 5-Fu group (72.79% vs 65.3%, Table 3).

The white blood cell count, total bilirubin and alanine aminotransferase (ALT) in the NS, CTS-Pasp and CTS-Pasp-5Fu groups were not significantly different (P > 0.05). Those in the 5-Fu group were higher than those in the other three groups (Table 4). There was no difference in creatinine among the four groups (P > 0.05).

The bone marrow inhibition effect in the 5-Fu group was significantly different from that in the other three groups (P < 0.001, Table 4, Figure 5).

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Figure 5 Bone marrow cell colonies (≥ 30 nucleated cells constituted one colony) in four groups (× 100). The growth of bone marrow cell colonies in the normal saline group (A), CTS-Pasp group (D) and CTS-Pasp-5Fu group (C) was vigorous, while the cell colonies in the 5-Fu (B) group was sparse. It demonstrated the inhibition effects of 5-Fu used alone. The marrow cell count indicated that CTS-Pasp-5Fu could not suppress the growth of bone marrow cells.

Nucleated cells ≥ 30 constituted one bone marrow cell colony[16] in the four groups (Figure 5). The growth of bone marrow cell colonies in the NS, CTS-Pasp and CTS-Pasp-5Fu groups was vigorous, while that in the 5-Fu group was sparse. CTS-Pasp-5Fu did not suppress the growth of bone marrow cells.

Table 5 shows the histological change in the four groups after treatment. The tumor cells in the NS and CTS-Pasp groups aligned regularly with slight inflammatory cell infiltration. The tumor cells in the CTS-Pasp-5Fu group were swollen with degeneration, necrosis and inflammatory cell infiltration which was less severe than in 5FU group. The damage liver in the 5-Fu group was more severe than that in the CTS-Pasp-5Fu group.

5-Fu and CTS-Pasp-5Fu down-regulate the Bcl-2 expression and up-regulation of the Bax expression were observed in the 5-Fu and CTS-Pasp-5Fu groups, but not in the CTS-Pasp group and NS group. The accommodate effect of CTS-Pasp-5Fu was more obvious than that of 5-Fu (Table 6, Figures 5 and 6).

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Figure 6 Immunohistochemical atlas of Bcl-2 and Bax expression in four groups (× 200). Bax expression is shown in normal saline group (A), CTS-Pasp group (B), 5-Fu group (C) and CTS-Pasp-5Fu group (D). Bcl-2 expression is shown in normal saline group(E), CTS-Pasp group (F), 5-Fu group (G) and CTS-Pasp-5Fu group (H). The masculine express of Bax in was CTS-Pasp-5Fu group was more obvious than that in the 5-Fu group. CTS-Pasp-5Fu and 5-Fu could up-regulate Bax expression compared with NS. The masculine expression of Bcl-2 in the CTS-Pasp-5Fu group was obviously lower than that in the other three groups. Both CTS-Pasp-5Fu and 5-Fu could both down-regulate the Bcl-2 expression.

Figure 6 shows the expression of the apoptotic proteins Bcl-2 and Bax in the four groups. The masculine expression of Bax in the CTS-Pasp-5Fu was more obvious than that in the 5-Fu group. CTS-Pasp-5Fu and 5Fu could up-regulate Bax expression. The masculine expression of Bcl-2 in the CTS-Pasp-5Fu group was obviously lower than that in the other three groups. The difference in the expression of Bax and Bcl-2 between NS group and CTS-Pasp group was not significant (Figure 6).

5-Fluorouracil (5-Fu) is often used as an anti-tumor agent in the treatment of gastrointestinal cancer. However, it often causes inconvenience of patients because of its short plasma half-life and requires continuous infusion (CI) schedules. So it is important to investigate and develop new drugs which can reduce the side efforts of 5-Fu.

Chitosan (CTS) has received more attention recently in the pharmaceutical field for a wide range of drug-delivery applications. Polyaspartic acid is a kind of a new biodegradable, innocuous and friendly environmental bio-organic polymer, recognized as a green material, and widely used in the areas such as agriculture, medicine, commodity, water treatment, etc.

In this study, the authors successfully synthesized chitosan-polyaspartic acid-5 fluorouracil (CTS-Pasp-5Fu) nanoparticles and investigated their anti-carcinoma effect and toxicity to overcome the disadvantage of 5-Fu in gastric carcinoma mouse model. The results show that the tumor inhibition rate of CTS-Pasp-5Fu nanoparticles was much higher than that of 5-Fu alone, and the inhibition on bone marrow was alleviated efficiently. Thus, it may be used as a safe and effective novel anti-tumor preparation with profound prospects in clinical application.

Based on the results of this study, it is supposed that CTS-Pasp-5Fu nano-particles may substitute 5-Fu in the future treatment of gastrointestinal cancer.

This study focused on the preparation of CTS-Pasp-5Fu nanoparticles and investigation of its anti-carcinoma effect and toxicity to overcome the disadvantage of 5-Fu in gastric carcinoma mouse model. The data demonstrate that CTS-Pasp-5Fu appears to offer comparable efficacy while providing a more convenient schedule and showed insight into the novel oral fluoropyrimidine.