In vivo and in vitro effects of Bletilla striata polysaccharide-loaded paclitaxel nanoparticles on human gastric cancer cells

Purpose: To investigate the in vivo and in vitro effects of paclitaxel nanoparticle (PTX)-loaded Bletilla striata polysaccharide (BSP) on human gastric cancer cells. Methods: Mice weighing 13 to 17 g and aged 4 to 6 weeks, were inoculated with human gastric gland cancer cell line (MKN45), and randomly assigned to five groups: control group, PTX-1 (10 mk/kg) group; PTX-2 (15 mg/kg) group, BSP-PTX-1 (10 mg/kg) group, and BSP-PTX-2 (15 mg/kg) group. The antiproliferative influence of BSP-PTX and its cellular target were determined using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence microscopy, respectively. Results: Inhibition of MKN45 cells was significantly higher in BSP-PTX group (88.24 %) than in PTX group (76.74 %, p < 0.05). More BSP-PTX entered the cells than PTX. Tumor inhibition was significantly low in PTX-1 group (37.58 %), relative to the BSP-PTX-I group (45.00 %, p < 0.5). In addition, tumor inhibition was significantly lower in PTX-2 group (52.35 %) than in BSP-PTX-2 group (69.80 %, p < 0.5). The weight gain of mice was lower in the PTX or BSP-PTX groups than in control mice, while the weight gain of mice in BSP-PTX-2 group (26.35 %) was significantly higher than that of PTX-2 group (19.43 %, p < 0.5). Conclusion: Bletilla striata polysaccharide-loaded paclitaxel nanoparticles enhance drug delivery, and effectively and safely exert anti-proliferative effect on MKN45 cells and in mice. Thus, these nanoparticles have good potential for development into anti-gastric cancer agents for clinical application.


INTRODUCTION
Since the beginning of this century, studies on carrier materials for functional anti-tumor drugs have received huge attention [1].Bletilla striata polysaccharide (BSP), a compound isolated from the rhizome of B. striata, confers protection on gastric mucosa.It can exert anti-tumor effects by inhibiting the growth and cell cycle arrest of human gastric cancer cells, human ovarian cancer cells (A2780) and liver cancer cells (HepG2) and possesses great biocompatibility and biodegradability properties [2,3].As a medical carrier material, it has great potential in clinical applications.Structurally, BSP contains numerous hydrophilic hydroxyl groups.However, it can be made into an amphipathic material with increased capability to trap insoluble anti-cancer drugs, and improved cellular targeting by the introduction of cholesteryl units [4].Although paclitaxel (PTX) exhibits good anti-cancer activity, its therapeutic effectiveness is poor due to its relatively poor water solubility.This study was aimed at investigating the in vivo and in vitro effects of paclitaxel nanoparticles loaded with Bletilla striata polysaccharide (BSP-PTX) on human gastric cancer cells.This study received approval from the Ethical Committee of our hospital (approval number: 20187891), and carried out according to the guidelines of the Declaration of Helsinki promulgated in 1964 as amended in 1996 [5].

Cell culture
Poorly-differentiated human gastric gland cancer cell line (MKN45) was cultured at 37 ℃ on DMEM in a 5 % CO 2 incubator.The cells were placed in thermostated water bath at 37 ℃ for 20 min to completely defrost, and then centrifuged.The supernatant was discarded, while DMEM was added to the pellets.The packed cells were dissociated into single cells and inoculated into culture bottles using a sterile sucker.The culture medium was further incubated at 37 o C in an atmosphere containing 5 % CO 2 , after which the cells were examined under an optical microscope.The culture medium was changed every 24 to 48 h.

MTT assay
This was carried out to assay the inhibitory effect of BSP-PTX on proliferation of MKN45 cells.After washing thrice in PBS, the cells were trypsinized and subsequently examined under inverted optical microscope within 1 min.After digestion of connective proteins among the cells, the pancreatic enzymes were discarded, followed by the addition of DMEM.Smears of the cells were prepared, mounted on a cover slide, viewed under a light microscope, and counted.The cells were maintained at a density of 2 × 10 4 /ml and seeded in 96-well plates.
The medium was removed, following incubation for 24 h, and different concentrations of the drugs ranging from 1.0 to 16.0 ng/mL were added.The culture medium was incubated for 96 h, after which the medium was discarded.A solution of MTT (100 uL) was added to each well and the wells were further subjected to incubation for 4 h under the same conditions as before, after which the supernatant was discarded.The medium in each well was replaced with 200 uL of DMSO and placed on an oscillator for 5 min, and subsequently examined under the microscope.The content of each well was read at 570 nm in a microplate reader, while the reference was read at 450 nm.The extent of inhibition (H) of cells was calculated as in Eq 1.
where Ac and At are the absorbance of control and treatment groups, respectively.

Determination of BSP-PTX target
The MKN45 cells were seeded in 96-well plates at a density 2 × 10 4 cell/well, and subjected to incubation for 24 h in a 5 % CO 2 incubator at 37 ℃, after which the supernatant was discarded.
The BSP-PTX and PTX were labelled with fluorescein isothiocyanate (FITC) injection for 3 h.The cells were thereafter rinsed thrice with PBS and viewed under a fluorescence microscope.

Transplantation of MKN45 cell and grouping
Mice weighing 13 to 17 g (mean weight = 15.2 g), aged 4 to 6 weeks (mean age = 5.1 weeks) were used.The MKN45 cells (2 × 10 7) were directly inoculated into the abdominal cavity of each mouse.After 3 weeks, the abdominal injection sites of mice were disinfected and their ascites excised with the aid of a 5-mL syringe.The mice were subsequently sacrificed by cervical dislocation and the excised ascites diluted with sterile saline solution (1:2 volume ratio) to a final concentration of 5 × 10 8 cells/mL.A portion (0.2 mL) was subcutaneously inoculated into the armpit of each mouse.
After 24 h of inoculation, the mice were allotted to 5 groups: control group, PTX-1 (10 mg/kg bwt) group, PTX-2 (15 mg/kg bwt) group, BSP-PTX-1 (10 mg/kg bwt) group, and BSP-PTX -2 (15 mg/kg bwt) group.The route of administration was intravenous (i.v.) injection, and administration was done 5 times at different time points: 1, 3, 5, 7, 9 days following inoculation.Weight, activity, capability and diet status were recorded on daily basis for 12 days.Then, following sacrifice via cervical dislocation, the mice were dissected, and the tumor tissues were excised and their weights were recorded.The extent of inhibition of tumor (TI) was calculated as in Eq 2. TI (%) = (1 -W)100 …….. (2) where W is mean weight of tumor in treatment group.

Statistical analysis
The results are presented as mean ± SEM.Comparison between groups was done with χ 2 test and Student's t-test.All analyses were done with SPSS ver.19.Values of p < 0.05 were taken as indicative of statistical significance.

Inhibition of cell proliferation
Higher inhibition of MKN45 cells was seen in BSP-PTX group (88.24 %), relative to PTX group (76.74 %) (p < 0.05).These results are shown in Table 1.Results of fluorescence microscopic examination showed that the drugs labelled with FITC entered the MKN45 cells.The amount of BSP-PTX that entered the cells was significantly higher than that of PTX.

Tumor inhibitory effects of PTX and BSP-PTX
Tumor Inhibition was significantly lower in PTX-I group (37.58 %) than in the BSP-PTX-I group (45.00 %) (p < 0.5).Tumor inhibition was also significantly lower in PTX-2 (52.35 %) than in BSP-PTX-2 (69.80 %) (p < 0.5).Significant reductions in mice weight were evident in the PTX/BSP-PTX groups, when compared with the control group, and weight change in mice in BSP-PTX-2 group (26.35 %) was significantly higher than that in the PTX-2 group (19.43 %) (p < 0.5).These results are shown in Tables 2 and  3.

DISCUSSION
The anti-cancer activity of TAX has become quite popular in recent times, and it is currently one of the anti-cancer drugs readily available in clinics [6].In 1992, the Food and Drug Administration (FDA) approved that TAX injection should be used for the treatment of metastatic ovarian cancer.Due to the poor solubility of PTX, polyoxyethylated castor oil is usually added to its solution as co-solvent [7].Polyoxyethylated castor oil causes allergic reactions and hyperlipidemia.Thus, it has become important to develop new formulations of PTX [8].In 2005, FDA approved that a combination of albumin and BSP-PTX injection should be used for the treatment of metastatic or recurrent breast cancer within 6 months after adjuvant chemotherapy [9].Studies have shown that this combination has higher bioavailability and safety than PTX [10].
Bletilla striata polysaccharide is a pectic polymer isolated from the rhizome of Bletillae and it confers protection on gastric mucosa [11].After fluorouracil white and polysaccharide microspheres, patients with primary liver cancer can release Fu in the body after hepatic artery embolization, which has long-acting, highefficiency and low toxicity [12].Compared with gelatin sponge, white and polysaccharide can significantly reduce the size of the tumor, the average time of recanalization and the half-year survival rate [13].It has advantages such as slow release, self-degradation and non-stimulatory effect, which make it a potential drug carrier material.Several studies have shown that the use of nanoparticles prepared with BSP can serve as good drug targets with features such as slow release, biocompatibility, self-degradation and reduced toxicity [14].Therefore, in the present study, the inhibitory effect BSP-derived taxol nanoparticles on human gastric cancer MKN45 cell growth was investigated.Taxol belongs to a class of insoluble anti-cancer drugs.Modification of BSP by dewatering converts it to an amphipathic carrier material which increases solvation of drugs, transport of drugs to target cells, and delay in drug release [15].In addition, BSP protects the gastric mucosa and enhances the release of prostaglandins.Some studies have suggested that BSP can be used for treating gastric cancer [16].
In the present investigation, higher inhibition of MKN45 cells was seen in BSP-PTX group, relative to the PTX group.This result suggests that BSP may change the pharmacological activity of PTX thereby conferring better antitumor activity on it.This property may be related to the packaging effect of BSP on PTX, which makes it easy for the nanoparticle form to enter cells.The amount of BSP-PTX that entered the cells was significantly higher than that of PTX.This may be an indication that BSP-PTX has a readier cellular target than PTX.
In this study, inhibition of tumor was significantly lower in PTX-I group than in the BSP-PTX-I group.The inhibition was also significantly lower in PTX-2 than in BSP-PTX-2.Lower weight gains were obtained in the PTX/BSP-PTX groups than that in control group, and weight gain was significantly higher in BSP-PTX-2 group than in the PTX-2 group.These results suggest that BSP-PTX may be less toxic to mice than PTX.The PTX-loaded nanoparticles appear to enter cells more easily than single PTX.Thus, PTXloaded nanoparticles have enhanced pharmacological functions.In addition, BSP has good biosafety.The polyoxy-ethylated castor oil contained in PTX injection easily causes multiple toxic reactions.Therefore, in terms of effectiveness and safety, compared with TAX, injection, BSP-PTX has treatment advantages.

Figure 1 :
Figure 1: Photomicrographs of MKN45 cells showing the targets of PTX and BSP-PTX.A: Control (no green fluorescence in the cells); B: PTX group (presence of light green fluorescence); C: BSP-PTX group (presence of dark green fluorescence)

Table 1 :
Anti-proliferative effect of different concentrations of TAX in the five groups

Table 3 :
Changes in weight of mice following treatment c p < 0.05, relative to PTX-2 group