Preparation of alginate oligosaccharide nanoliposomes and an analysis of their inhibitory effects on Caco‐2 cells

Abstract

The conditions were optimised for preparing Alginate oligosaccharide (AOS) nanoliposomes, and Caco‐2 cell experiments were carried out to examine their antitumour effects. The optimal formulation of AOS nanoliposomes was as follows: a phosphatidylcholine‐to‐cholesterol ratio of 5.12, AOS concentration of 8.44 mg/mL, Tween 80 concentration of 1.11%, and organic phase to aqueous phase ratio of 5.25. Under the above conditions, the experimental encapsulation efficiency was 65.84%, and the AOS nanoliposomes exhibited a small particle size of 323 nm. After Caco‐2 cells were treated with AOS liposomes and AOS for 24 h, AOS nanoliposomes inhibited the growth of Caco‐2 cells to a greater extent than AOS at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL (P  <  0.01). LDH leakage exhibited a concentration‐dependent increase following treatment with 0.5‐1 mg/mL AOS nanoliposomes, and the inhibitory effect of AOS nanoliposomes exhibited a more significant difference than AOS (P  <  0.01). Cells treated with 0.5 mg/mL and 1 mg/mL AOS nanoliposomes displayed a substantial and significant increase in activity compared with AOS (P  < 0.01). Based on these results, AOS nanoliposomes exerted a more significant effect on inhibiting Caco‐2 cell proliferation than AOS.

1 Introduction

Alginate oligosaccharide (AOS) is any short chain of sugar residues interconnected by glycosidic linkages [1]. AOS is regarded as a biocompatible, non‐toxic, non‐immunogenic, and biodegradable polymer, suggesting that it is an attractive candidate for biomedical applications [2]. AOS displays a wide range of physiological activities, such as anti‐aggregatory effects [3], anti‐inflammatory effects [4, 5, 6], immuno‐modulatory effects [7, 8], and neuroprotective effects [9]. In addition, AOS has great potential in preventing cancer cell growth because of its characteristics, such as safety and bioavailability. As shown in the study by Iwamoto et al. [7], AOS could retain its bioactivity and induce apoptosis in U937 human leukaemia cells. However, AOS must enter cancer cells before it inhibits cell growth. More efforts are needed to improve the AOS utilisation efficiency by developing colloidal delivery systems for cells, such as liposomes and micro or nanoparticles [10].

Liposomes are a type of vesicle with a phospholipid bilayer membrane that has been extensively reported to enhance the therapeutic efficacy of different drugs in the field of drug delivery systems. Nanoliposomes share the same advantages as nanoparticles, potentially improving the effectiveness of AOS when used as a delivery system. Nanoliposomes might enhance the stability of the encapsulated material by protecting it from environmental factors [11, 12, 13] and have been chosen as a prospective candidate drug delivery system. Developments in liposomal delivery systems (stealth liposomes) may facilitate the targeting of specific agents to cancer for treatment. These systems could be developed as platforms for future multi‐functional nanodevices that are tailor‐made for the combined early detection of cancer and delivery of functional drugs. On the other hand, nanoliposomes have been used as immunological adjuvants to promote both humoural and cell‐mediated immunity [14, 15]. Nanoliposomes targeting tumour tissues have already displayed excellent functions in some studies [13, 16]. Currently, no report has described the use of nanoliposomes as the delivery system to transport AOS into tumour cells. In this study, an AOS‐nanoliposome system was prepared using the reverse‐phase evaporation method. Furthermore, its effects on inhibiting the growth of Caco‐2 cells were inhibited.

2 Methods

2.1 Materials

AOS was obtained from Qingdao Bozhi Huili Biotechnology Company (Shandong, China). Phosphatidylcholine (PC) was obtained from Base Biotechnology Company (Hangzhou, China). Cholesterol (CH) was obtained from Aladdin Industrial Corporation (Shanghai, China). Caco‐2 cells were purchased from Meixuan Biological Science and Technology Co., Ltd. (Shanghai, China). The other chemicals were of reagent grade.

2.2 Preparation of AOS nanoliposomes

AOS nanoliposomes were prepared using the reverse‐phase evaporation method [17, 18]. Actually, specific amounts of PC and CH were dissolved in the chloroform‐diethyl ether, and AOS was dissolved in 3 ml of phosphate‐buffered saline (PBS) (pH 7.4). The organic phase was mixed with the aqueous phase for 5 min using probe sonication. The mixed product was placed in a round‐bottom flask that was directly attached to a rotary evaporator to evaporate the organic solvent at 38°C under vacuum and obtain a dry, uniform, and thin film on the inner wall of the flask. Finally, 30 ml of PBS containing Tween 80 was added to the flask, and the film and the PBS were sonicated for an additional 17 min to produce a homogeneous aqueous suspension.

2.3 Determination of the encapsulation efficiency (EE)

The EE was determined by calculating the concentration of AOS entrapped in nanoliposomes and free AOS in the aqueous phase using the centrifuge‐UV method. One millilitre of liposomal suspension with AOS was removed and centrifuged at 10,000 rpm for 30 min at 4°C to separate the encapsulated AOS from the aqueous phase. A suspension of the same volume was ruptured using a sufficient volume of ethanol, and the total amount of AOS was measured using spectrophotometric methods. The percentage of EE (EE%) was calculated using the following equation:

$$\mathrm{EE}\text{\%}=\frac{E_2-E_1}{E_2}\times 100\text{\%}$$

where E ₁ is the amount of free AOS and E ₂ is the total amount of AOS in 1 ml of nanoliposomes.

2.4 Particle size

The mean vesicle size of the liposomes was examined using a laser scattering method (Nano ZS 90, Malvern, UK). The determination was repeated three times per sample.

2.5 Experimental design and optimisation of the AOS liposome preparation

Response surface methodology (RSM), a generic method for optimisation, was applied to optimise the formulation of AOS liposomes. Based on the results of single‐factor experiments and our previous studies, four factors, the PC/CH ratio (X ₁), AOS concentration (X ₂), Tween 80 concentration (X ₃), and organic phase to aqueous phase ratio (X ₄), were selected to optimise the conditions used to prepare AOS liposomes. The optimisation strategy was designed based on a four‐factor‐three coded level Box–Behnken design (BBD) with a total of 29 experimental runs. When considering the feasibility of preparing liposomes, the ranges of four factors were determined as follows: PC/CH (3–6, w/w), AOS concentration (6–11, w/v), organic phase to aqueous phase (3–7, v/v), and Tween 80 concentration (0.5–1.5, w/v) (Table 1). The response was related to the selected variables using a second‐order polynomial model. The generalised model proposed for the response was calculated using the following equation:

$$Y_i={\beta }_0+\sum _i{\beta }_i{X}_i+\sum _i{\beta }_{ii}{X}_i^2+\sum _{i\ne j}{\beta }_{ij}{X}_i{X}_j$$

where Y_i is the response, β ₀ is the constant coefficient, β_i is the linear coefficient, β_ii is the squared coefficient, β_ij is the interaction coefficient, and X_i and X_j are the coded values of independent variables [19]. Multivariate regression analyses and analysis of variance (ANOVA) were used to determine and verify the statistical significance. The regression coefficient R ², adjusted R ², coefficient of variation F ‐value, and lack of fit were used to evaluate the significance of the model. Moreover, the adequacy of the model was assessed to account for coefficient of variation and adjusted R ² value.

Table 1.

Factors and levels used in the three‐level, four‐variable response surface experiments

Independent variables	Symbols	Code levels
		−1	0	1
PC/CH (w/w)	X 1	3	4.5	6
AOS concentration (w/v)	X 2	6	8.5	11
Tween 80 concentration (w/v)	X 3	0.5	1	1.5
organic phase to aqueous phase ratio	X 4	3	5	7

2.6 Cell culture

Caco‐2 cells were grown in Dulbecco's modified eagle medium (DMEM) (Sangon Biotech, Hangzhou, China) supplemented with 10% foetal bovine serum, a 1% non‐essential amino acid solution, and a 1% penicillin/streptomycin solution in a humidified atmosphere of 5% CO₂ at 37°C. Cells were passaged every three days to maintain rapid growth. Upon reaching 80% confluence, cells were transferred to 96‐well plates for the next subculture. When the monolayer of cells was formed after 24 h, cells were treated with different concentrations of AOS liposomes that contained an AOS concentration equal to the AOS group. The three replicates of 96‐well plates were included in each experiment.

2.7 Methyl thiazolyl tetrazolium (MTT) assay

Cell viability was measured using the MTT reduction assay [13]. Monolayers of cells were treated with 0.0625, 0.125, 0.25, 0.5, or 1 mg/ml of AOS liposomes or AOS. After the 24 h treatment, serum‐free medium containing 0.5 mg/ml MTT was added to the cells, mixed and incubated for 4 h in a CO₂ incubator. The culture medium in each well was carefully aspirated to avoid disturbing the cell monolayer. Then, 100 μl of formazan solubilisation solution of Dimethyl sulfoxide (DMSO) was added to each well and gently mixed for 10 min on a shaker until complete dissolution. By measuring the absorbance of the enzymatic reduction of the yellow tetrazolium MTT assay to a purple formazan product at 490 nm, cell viability was enzymatically assessed with the MTT assay using a spectrophotometer [20, 21]. The percentage of cell viability was calculated using the following equation:

$$\mathrm{Cell}\mathrm{viability}\left(\text{\%}\right)=\frac{A_1-A_0}{A_2-A_0}\times 100$$

where A ₁ is the absorbance of formazan measured at 490 nm in the treated group and A ₂ is the absorbance of formazan measured at 490 nm in the control group.

2.8 Lactate dehydrogenase (LDH) assay

After the plates had been incubated in a humidified incubator for 24 h, cells were seeded (10,000 cells per well) in 96‐well plates. Cells were treated with different concentrations of AOS nanoliposomes and AOS for 24 h. Negative control cells were cultured in medium without the AOS compounds, positive control cells were cultured in medium with LDH to release the reagent, and the medium of blank groups did not include any cells. Media from each well (test, positive control, negative control, and blank wells) were centrifuged at 1000 rpm for 5 min, and 120 μl of the supernatant was transferred to the corresponding well of a 96‐well test plate. After 60 μl of the working reaction mixture was added to each well, the plates were placed on a shaking table and incubated in the dark at room temperature for 30 min. The absorbance was recorded at 490 nm using a microplate reader, and LDH leakage was calculated using the same formula as used for the MTT assay.

2.9 Cell proliferation

Cell counting kit‐8 (CCK‐8) is a sensitive assay for the determination of cell viability in cell proliferation and cytotoxicity assays. After plates had been incubated in a humidified incubator for 24 h, cells were seeded (10,000 cells per well) in 96‐well plates. Monolayers of cells were treated with 0.0625, 0.125, 0.25, 0.5, or 1 mg/ml of AOS liposomes or AOS for 24 h. After the culture medium was removed from each well, the treated cells were washed twice with PBS. Then, 90 μl of serum‐free DMEM medium and 10 μl of CCK‐8 solution were added to each well, and the culture was incubated for an additional 1.5 h at 37°C. The absorbance was measured at 450 nm using a microplate reader. Three parallel cultures were used to assess the viability of cells in each sample, and cell viability was calculated using the same formula described for the MTT assay.

2.10 Statistical analysis

Data from the optimisation of the AOS nanoliposome preparation were analysed using Design‐Expert 8.0 software and a second‐order polynomial equation, as well as an ANOVA of the quadratic regression model; the optimal conditions are shown. The results are presented as the means ± standard deviations of three independent experiments, and P  > 0.05, P  < 0.05, and P  < 0.01 were, respectively, regarded as not significant, significant, and extremely significant differences.

3 Results and discussion

3.1 Statistical analysis and fitting of the optimised method for preparing AOS nanoliposomes

Twenty‐nine experimental runs were employed to optimise the four individual parameters using the BBD. The experimental conditions used to determine the EE and particle size of AOS nanoliposomes are shown in Table 1, according to the factorial design, and the results are shown in Table 2. A regression analysis and ANOVA were used to fit the model and examine the statistical significance of the terms. The estimated regression coefficients were calculated for each response variable, along with the corresponding R ² value, adjusted R ² (adj‐R ²) value, F ‐value, and P ‐value for the lack of fit. In this study, the R ² values for the response variables EE and particle size were 0.9711 and 0.9686, respectively; values that were >0.80 indicated that the regression models explained the reliability. Moreover, the addition of a variable to the model would always increase the R ² value, regardless of whether the additional variable was statistically significant, and an adj‐R ² value was used to evaluate the adequacy of the model. The R ² and adj‐R ² values for the model were not clearly different and indicated that non‐significant terms were not reflected in the model. Normally, the lack of fit indicates that the model does not represent the data in the experimental domain, for which points are not present in the regression curve. The lack of fit of EE and particle size were 0.9281 and 0.1650, respectively, which were not significant (P  > 0.05) for the response surface model and implied that the established model accurately represented the relevant data.

Table 2.

Design and results of the response surface experiments for EE and size

Run	EE, %	Size, nm	Run	EE, %	Size, nm
1	57.29	422.1	16	57.33	524.3
2	61.81	397.7	17	63.66	518.4
3	58.08	489.0	18	66.07	317.1
4	59.27	429.5	19	49.23	491.0
5	53.74	520.8	20	52.07	548.9
6	61.86	473.5	21	48.8	413.5
7	65.39	347.6	22	54.19	400.0
8	55.37	473.7	23	65.28	325.9
9	60.69	399.7	24	49.76	356.6
10	58.81	524.0	25	51.32	385.3
11	61.26	417.4	26	61.48	323.9
12	65.69	327.6	27	59.12	441.3
13	54.93	430.4	28	48.03	384.6
14	57.89	528.6	29	52.65	443.9
15	48.43	448.0	—	—	—

3.2 Encapsulation efficiency

The regression coefficients were calculated, and the response variable and the test variables were related by the following second‐order polynomial equation:

$$\begin{array}{rl}Y& =64.78+4.42X_1+0.71X_2+1.26X_3+2.21X_4\\ & -1.47X_1{X}_2-0.31X_1{X}_3-0.29X_1{X}_4\\ & -3.32X_2{X}_3-1.11X_2{X}_4-3.15X_3{X}_4\\ & -3.44X_1^2-6.64X_2^2-2.27X_3^2-5.92X_4^2\end{array}$$

P ‐values served as a tool to examine the significance of each coefficient; a smaller p ‐value represented a more significant corresponding coefficient [15]. P ‐values of <0.05 indicated that model terms were significant.

The linear coefficients (X ₁, X ₃, and X ₄), quadratic term coefficients ($X_1^2$, $X_2^2$, $X_3^2,\mathrm{and}{X}_4^2$) and interaction coefficients (X ₁ X ₂, X ₂ X ₃, and X ₃ X ₄) were significant (P  <  0.01), but the other term coefficients were not significant (P  >  0.05). The effects of the independent variables on AOS nanoliposomes are shown in Fig. 1. As shown in Fig. 1 a, the EE was enhanced as the PC‐to‐CH ratio and AOS concentration increased. The increased EE might be due to the easier encapsulation of AOS into the vesicles as the AOS concentration increased, and CH modulated the order of mobility of lecithin in the lipid bilayer, potentially reinforcing the membrane stability [22]. As shown in Fig. 1 b, the increase in the Tween 80 concentration and organic phase to aqueous phase ratio enhanced the EE of AOS nanoliposomes. Due to the better availability of lipophilic moieties, the higher EE might be attributed to the improved density of the liposome surface, which would accommodate a greater amount of the encapsulated material [23].

Fig. 1.

Effect of interactions between different factors on the EE

(a) Effects of the PC‐to‐CH ratio and AOS concentration (Tween 80 concentration = 1.11 mg/ml and organic phase to aqueous phase ratio = 5.25), (b) Effects of the Tween 80 concentration and organic phase to aqueous phase ratio (PC‐to‐CH ratio = 5.12 and AOS concentration = 8.44 mg/ml)

3.3 Particle size

In terms of the sum of squares, the importance of independent variables for yield was ranked in the following order: AOS concentration > Tween 80 concentration > PC‐to‐CH ratio > organic phase to aqueous phase. The variation in size with AOS concentration and PC‐to‐CH ratio is presented in Fig. 2 a. As phospholipids constituted the liposome membrane and the PC concentration exerted a direct effect on the particle size of the liposomes [19], the particle size of AOS nanoliposomes decreased as the PC concentration decreased. The effects of the AOS concentration and organic phase to aqueous phase ratio on the nanoliposome size are presented in Fig. 2 b. Additionally, different Tween 80 concentrations and organic phase to aqueous phase ratios have been reported to distinctly affect the particle size and dispersion of prepared liposomes.

Fig. 2.

Response surface plot of the effects of independent variables on the size of AOS nanoliposomes

(a) Effects of AOS concentration and PC‐to‐CH ratio (Tween 80 concentration = 1.11 mg/ml and organic phase to aqueous phase ratio = 5.25), (b) Effects of the Tween 80 concentration and organic phase to aqueous phase ratio (PC‐to‐CH ratio = 5.12 and AOS concentration = 8.44 mg/ml)

3.4 Optimisation

Based on the effects of PC/CH, the AOS concentration, Tween 80 concentration, and organic phase to aqueous phase ratio on the formulation of AOS nanoliposomes, the ranges of each independent variable were optimised to prepare AOS nanoliposomes with the highest EE. The optimal formulation was produced using the following parameters: a PC‐to‐CH ratio of 5.12, an AOS concentration of 8.44 mg/ml, a Tween 80 concentration of 1.11%, and an organic phase to aqueous phase ratio of 5.25. The particle size was determined for the AOS nanoliposomes prepared using the optimised method, and the size distribution is shown in Fig. 3. The conditions yielded the highest EE (66.25 ± 1.23%) and a low value for the particle size (323 ± 3 nm). The determined values were similar to the predicted values, verifying that we utilised the optimised preparation conditions. Based on the experimental results, the model used in this study should be regarded as the optimal conditions for preparing AOS nanoliposomes, consistent with the criteria for nanoliposomes reported in previous studies [11, 12].

Fig. 3.

Plot of the particle size distribution of optimised AOS nanoliposomes versus intensity. The AOS nanoliposomes exhibited a small particle size of 323 nm

3.5 Evaluation of the effects of AOS and AOS nanoliposomes on Caco‐2 cells

The results of the MTT assay revealed a concentration‐dependent effect of AOS and AOS nanoliposomes on Caco‐2 cells (Fig. 4). After cells were exposed to 0.0625, 0.125, 0.25, 0.5, or 1 mg/ml of AOS or AOS nanoliposomes for 24 h, the observed percentages (%) of viable cells (relative to control) were 95.96 and 91.63%, 87.91 and 78.25%, 76.54 and 69.22%, 65.47 and 53.23%, and 54.60 and 44.69%, respectively. A statistically significant difference in the percentages of viable cells was observed between cultures treated with AOS and AOS nanoliposomes. Compared with AOS, AOS nanoliposomes exerted a more substantial effect on decreasing the viability of Caco‐2 cells (P  < 0.01).

Fig. 4.

Viability of Caco‐2 cells after treatment with AOS or AOS nanoliposomes for 24 h, as determined by the MTT assay. The data are reported as the mean values ± standard deviations of three replicates (*P < 0.01, compared with the Ctrl group, #P < 0.01, compared with the AOS group)

LDH is an oxidoreductase that catalyses the interconversion of lactate and pyruvate and often impairs cell membrane integrity by inducing cell apoptosis or necrosis [24]. LDH leakage is regarded as a useful index of cell viability and the loss of membrane integrity. After Caco‐2 cells were treated with AOS or AOS nanoliposomes for 24 h, LDH leakage increased in a concentration‐dependent manner, and 0.5–1 mg/ml of AOS nanoliposomes obviously reduced the viability of tumour cells. The effect of AOS nanoliposomes was more significant (P  < 0.01) than the effect of AOS (Fig. 5).

Fig. 5.

LDH leakage from Caco‐2 cells cultured in the presence of AOS or AOS nanoliposomes for 24 h. The data are reported as the mean values ± standard deviations of three replicates (*P < 0.01, compared with the Ctrl group, #P < 0.01, compared with the AOS group)

In the CCK‐8 assay, Caco‐2 cells were treated with AOS or AOS nanoliposomes (0.0625–1 mg/ml) for 24 h. The proliferation of cells treated with 0.125, 0.5, and 1 mg/ml AOS nanoliposomes was reduced by 79.63, 52.04, and 40.72%, respectively, compared with AOS‐treated cells (Fig. 6). The proliferation of cells treated with AOS nanoliposomes at concentrations of 0.5 and 1 mg/ml was extremely significantly different from cells treated with the same concentrations of AOS (P  < 0.01). Thus, AOS nanoliposomes inhibited tumour cell proliferation to a greater extent than AOS.

Fig. 6.

Proliferation of Caco‐2 cells treated with AOS and AOS nanoliposomes for 24 h, as measured using the CCK‐8 assay. The data are reported as the mean values ± standard deviations of three replicates (*P < 0.01, compared with the Ctrl group, #P < 0.01, compared with the AOS group)

AOS has been reported to modulate the growth of cancer cells [7, 25], and AOS enhances its anticancer activity by inducing the production of cytotoxic cytokines in human mononuclear cells [7]. In this study, the prepared AOS nanoliposomes exhibited a more obvious effect on Caco‐2 cell viability than the same concentration of AOS. The ability to facilitate the uptake and cytoplasmic delivery of drugs to specific tissues and cells has rendered liposomes a versatile delivery vehicle with numerous potential advantages [19, 26]. Consistent with these findings, AOS nanoliposomes carried more AOS into the Caco‐2 cells and increased the efficiency and effectiveness of the AOS drug.

4 Conclusions

AOS nanoliposomes were prepared under the optimal conditions of a PC‐to‐CH ratio of 5.12, AOS concentration of 8.44 mg/ml, Tween 80 concentration of 1.11%, and organic phase to aqueous phase ratio of 5.25. The prepared AOS nanoliposomes significantly inhibited the growth of Caco‐2 cells by promoting the ready entry of AOS into cells and thus represent a potentially suitable system for prospective application in controlling tumour cell growth.