MATERIALS AND METHODS

Materials: Polyvinyl Alcohol (PVA), Wheat Germ Agglutinin (WGA), Carbodiimide (EDAC) N-hydroxysuccinimide (NHS), Folin-Ciocalteu's phenol Reagent, poly (lactic-co-glycolic acid) (PLGA), matrine (MT) and tetrazolium salt reagent (MTT) were purchased from companies with good quality and reputation.

HT-29 colon cancer cell lines (Shanghai Institute of Biochemistry and Cell Biology), NCM-460 human normal colonic epithelial cell line (Guangzhou Jini Ou Biotechnology Co., Ltd.), Annexin V-FITC/PI Apoptosis Detection Kit (Beijing Kang Century Biotech Co., Ltd.), DNA content detection kit (Nanjing KGI Biological Technology Development Co., Ltd.) and Dil red fluorescent membrane probe (Bi Yun Tian Biotechnology Co., Ltd), etc. were used. The entire research was conducted at Henan University of Traditional Chinese Medicine in the period from December, 2017-January, 2019.

Preparation of WGA modified matrine nanoparticles (WGA-MT-NPs): WGA-MT-NPs were prepared using the double emulsification-solvent evaporation method followed by the carbodiimide method as previously described^15,16. Briefly, after the hot water solution of MT was dropped into the acetone solution of PLGA under magnetic stirring, the resulting emulsion was injected rapidly into the water solution of PVA under stirring condition. Subsequently, the resulting solution wascontinuously stirred to remove acetone and filtered to obtain MT nanoparticles (MT-NPs).After PBS solution of EDAC and NHS added into the MT-NPs solution to activate for 4 hrs, EDC and NHS were removed by centrifugation. Then PBS solution of WGA was added to modify for 14 hrs, WGA-MT-NPs were obtained by centrifugation and wash to remove free lectin and lyophilized.

Characterization: HPLC and nano-laser particle size analyzer were used to measure encapsulation efficiency, average particle size and potential value, respectively. The folin-phenol method was used to measure the unbound WGA lectin as per manufacturer’s instructions (Sigma):

And transmission electron microscopy (TEM) was used to determine the image of WGA-MT-NPs.

Interactions between WGA-MT-NPS and HT-29 cells as well as NCM460 cells: The affinity of nanoparticles towards colon cells was measured using fluorescein isothiocyanate (FITC) labeled WGA, according to the method reported by Tammam et al.¹⁷ with modification. Briefly, after HT-29 cells or NCM4460 cells (1×10⁵ mL^–1) were seeded and cultured for 24 hrs, WGA-MT-NPs solution (200 μg mL^–1 of MT) was added. Cells were then incubated for 25 min, washed with PBS, stabilized for 10 min with 4% paraformaldehyde at room temperature, washed again with PBS and immediately added the plasma membrane fluorescent dye Dil 500 μL pre-balanced at room temperature to incubate for 15 min. Discarding Dil, cells were washed and imaged with confocal microscopy.

Effect on HT-29 human colon cancer cell proliferation: HT-29 cells (1×10⁵ mL^–1) were seeded and cultured in a 96-well plate for 24 hrs. WGA-MT-NPs and MT-NPs with MT concentrations of 25, 50, 100, 200 and 400 μg mL^–1 were separately added. After culturing for 24, 48 and 72 hrs, the MTT method was used to detect cell proliferation as described previously¹⁸.

Effects on HT-29 human colon cancer cell cycle and apoptosis: HT-29 cells (2×10⁵ mL^–1) were seeded and cultured for 24 hrs. WGA-MT-NPs or MT-NPs were added at final concentration 100, 150 and 200 μg mL^–1 of MT. After 48 hrs, cells were digested with 0.25% trypsin, washed with 4°C pre-cooled PBS buffer. Then, cells were treated according to manufacturer’s instructions for detecting cell cycle and cell apoptosis by flow cytometry. The apoptotic rate was thesumofthe percentageof earlyandlate apoptosis.

Statistical analysis: Statistical analysis was performed by SPSS19.0 software using the one way ANOVA, p<0.05 and <0.01 represented significant and extremely significant differences, respectively.

RESULTS

MT encapsulation efficiency and WGA modification rate: The encapsulation efficiency of WGA-MT-NPs was 89.99±5.08% according to the result of HPLC (Fig. 1a). And, according to the standard curve of WGA: y = 4.789×10^–4 x+0.7107, R = 0.9996 (Fig. 1b), the modifying efficiency of WGA for MT-NPs was determined as 87.57±8.61%.

Fig. 1(a-f):	HPLC chromatogram, (a, b) WGA-MT-NPs and standard curve of WGA, (c, d) MT-NPs size distribution and zeta potential and (e,f) WGA-MT-NPs size distribution and zeta potential

Fig. 2(a-b):	TEM images of (a) MT-NPs and (b) WGA-MT-NPs

Fig. 3(a-f):	Confocal images of WGA-MT-NPs affinity towards HT-29 cancer cells and NCM460 normal cells
	a-b: FITC-WAG, c-d: Dil red fluorescent membrane, e-f: Merged channels

Size distribution, zeta potential and morphology: As shown in Fig. 1c-f, the sizes and potential of MT-NPs and WGA-MT-NPs were 102.93±5.53 nm, -12.73±0.68 mV and 220.10±13.80 nm, -10.50±0.70 mV, respectively. TEM images further revealed their morphology. The MT-NPs particles were a distinct spherical or near-spherical shape (Fig. 2a). However, WGA-MT-NPs (Fig. 2b) were significantly changed after modified by WGA, became irregular with some viscous material attached to MT-NPs core.

Higher affinity between WGA-MT-NPs and HT-29 cells: The Fig. 3 reveals the affinity of WGA-MT-NPs. After the FITC-WGA conjugates were used to prepare WGA-MT-NPs, HT-29 cells had obvious green fluorescence, while NCM460 normal colon cells only had very little green fluorescence. The overlap of the FITC green fluorescence and the red fluorescence of Dil, a lipophilicmembranestain, in HT-29 cells had evident co-localization, showing that WGA-MT-NPs had a significant stronger affinity towards HT-29 cells membrane compared with NCM460 cells.

Effects of WGA-MT-NPs on HT-29 cells proliferation: The cell inhibition in vitro is shown in Fig. 4. Within the concentration at 25-100 μg mL^–1 of MT, the inhibition rate of WGA-MT-NPs on HT-29 cells were 3-6 times compared to that of MT-NPs. Withan increaseof MT concentration to 200 μg mL^–1, WGA-MT-NPs inhibition rate had almost reached its maximum, which was up to 81.6% at 48 hrs.

Fig. 4:	WGA-MT-NPs enhanced the growth inhibition of MT-NPs in HT-29 cells
	The effect on HT-29 human colon cancer cell proliferation was detected by MTT method

Whereas the inhibition rate at 48 hrs was only 41.6% even if the MT-NPs concentration reached 400 μg mL^–1. The result suggested that with the help of the WGA, the effect of WGA-MT-NPs at low concentrations nearly equal at high concentrations of MT-NPs.

Effect of WGA-MT-NPs and MT-NPs on HT-29 cell cycle and apoptosis: Results in Fig. 5 show the effects of the nanoparticles on the HT-29 cell cycle. Compared with the control group, treatment with 100, 150 and 200 μg mL^–1 both MT-NPs and WGA-MT-NPs were able to significantly increase the cells at the G1 phase and decrease the cells at S phase (p<0.05 and <0.01) with a dose-dependent effect. However, no statistically significant differences were observed between the WGA-MT-NPs group and the MT-NPs group.

As shown in Fig. 6, both MT-NPs and WGA-MT-NPs can induce HT-29 cell apoptosis. Compared with the control group, the 100, 150 and 200 μg mL^–1 of MT-NPs groups caused significant apoptosis with an apoptotic rate of 19.06, 22.73 and 30.20%, respectively (p<0.01). The corresponding WGA-MT-NP caused more severe apoptosis with an apoptotic rate of 26.82, 38.30 and 55.70%, respectively. The significant differences were found between the WGA-MT-NPs group and the same concentration MT-NPs group at 150 and 200 μg mL^–1.

DISCUSSION

The modification of WGA was confirmed by the increase of the average diameter of nanoparticles (Fig. 1c, e) and the change of shape from near-spherical to non-uniform (Fig. 2a, b), which may be due to WGA grafting on the surface of MT-NPs, its hydrophilic properties can make the protein’s macromolecular chains extend to the water phase and cause the hydrated radius of the nanoparticles to increase. Increased size after WGA modification in the present study is in agreement with Liu et al.¹⁹ report.

WGA-MT-NPs had better affinity on HT-29 cells, rather normal colon cells (Fig. 3a, b). It was reported that the cell surface of colon cancer epithelial cells, such as Caco-2 and HT-29, had a large number of glycoproteins, while there was an apparent lack of these glycoproteins on normal colon cells^20,21, thus CRC could be successfully distinguished from normal cells by targeting this glycosylation with affinity capillary electrophoresis²². Our result also accounts for targeting glycoproteins, the binding sites of WGA, could recognize colon cancer cells.

Protein glycosylation is the attachment of carbohydrate to the amino acid residue of the protein backbone^23,24. O-GlcNAcylation in which N-acetyl glucosamine (GlcNAc) is added to the serine or threonine residue of protein, N-linked and O-linked modifications are the commonly occurring types of glycan modifications of protein²⁵. Altered mucin expression in conjunction with differential glycosylation has been strongly associated with malignant pathologies of colon²⁶. The aberrant glycosylation, such as an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans and O-GlcNAcylation, have been reported in CRC²⁰. Also, WGA can bind GlcNAc-related oligosaccharides and has applications as a commercial reagent to detect glycans containing GlcNAc modified residues²⁷ and WGA was used to determine the mucin secreted by colon carcinoma Caco-2 and mucus-producing HT29-MTX-E12 cell monolayers²⁸. And, a lectin-based reverse-phase protein microarray method had been developed for screening the glycosylation pattern of receptors isolated from thepatientsofcoloniccarcinoma²⁹.

Fig. 5(a-g):	Effect on HT-29 human colon cancer cell cycle
	a: Control, b-d: 100, 150 and 200 μg mL–1 of MT-NPs, e-g: 100, 150 and 200 μg mL–1 of WGA-MT-NPs

Fig. 6:	Effect on HT-29 colon cancer cell apoptosis
	Percent of apoptosis cells included the early and late apoptosis cells. #p<0.05, ##p<0.01 vs the control group, **p<0.01 vs the same concentration of MT-NPs group

All of these studies may further explain the mechanisms of WGA targeting CRC. These mechanisms may contribute to the targeting function of WGA-MT-NPs.

Modification with WGA significantly increased the growth inhibitory activity of the corresponding MT-NPs group at the same concentration (Fig. 4). In accordance with our results, Pandey et al.³⁰ also reported the WGA-conjugated polylactic-co-glycolic acid nanoparticles of 5-fluorouracil could significantly inhibit the growth of colon cancer cells compared to the non-conjugated NPs and pure drug. Also, recent study has shown that glycosylation and glycan interactions affect clathrin-independent endocytosis³¹. And, the lipid nanoparticles of bufalin with WGA could also increase bufalin's uptake by colon cancer CaCo-2 cells in vitro³². These results suggested that the improved inhibition effects of MT-NPs by WGA modification may be related to the uptake.

Both WGA-MT-NPs and MT-NPs were able to hinder DNA synthesis and induce apoptosis in HT-29 cells (Fig. 5 and 6). However, compared with the same concentration MT-NPs, WGA-MT-NPs significantly enhanced the apoptosis of HT-29 cells (Fig. 6). This may be because MT can inhibit DNA synthesis and induce apoptosis in HT-29 cells^3,33 and with the help of WGA, WGA-MT-NPs enabled more cells to apoptosis. In similar to our results, WGA-EF-NPs (5-fluorouracil and epigallocatechin-3-gallate co-loaded in wheat germ agglutinin-conjugated nanoparticles) exhibited superior anti-tumor activity and pro-apoptotic efficacy compared to the drugs nanoparticles without WGA decoration³⁴. Therefore, the significantly increased apoptosis in HT-29 cells may play a role in WGA-MT-NPs significantly enhancing the antitumor effect of MT-NPs.

CONCLUSION

WGA-MT-NPs significantly increased the antitumor effect of MT-NPs via stronger affinity towards HT-29 cells, higher promotion of apoptosis and maintenance of inhibiting proliferation. Thus, WGA-MT-NPs may provide a promising new strategy for anti-colon cancer. The potential of WGA-MT-NPs for the effective treatment of CRC is worthy of further study in vivo.

SIGNIFICANCE STATEMENT

This study discovered the targeting function and the increased apoptosis effect of WGA modifying matrine nanoparticles, which can be beneficial for improving the anti-colon cancer efficiency of matrine. This study will help the researchers to uncover the critical areas ofcoloncancer treatment with MT’s nano-pharmaceutics that many researchers were not able to explore. Thus a new therapy strategy of MT on colon cancer may be arrived at.

ACKNOWLEDGMENTS

This study was supposed by the Scientific and Technological Projects of the Science and Technology Department of Henan Province (NO: 132102310267). We would like to give our thanks to Professor Sugai Yin in Henan University of TCM and Dr. Jie Zhou in Zhengzhou University for their guidance in technologies.