Preparation and characterization of scutellarin loaded on ultradeformable nano-liposomes scutellarin EDTMP (S-UNL-E) and in vitro study of its osteogenesis

Abstract

The present research aimed to elucidate a convenient, safe and economic approach to induce the growth of endogenous bone tissue and bone regeneration. S-UNL-E was prepared using reverse-phase evaporation, and scutellarin encapsulation was subsequently compared. Meanwhile, the optimal preparation scheme was developed using an orthogonal method, and the particle size was determined using laser light scattering. In osteoblasts cultured in vitro, methyl thiazolyl tetrazolium (MTT), alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic effects of S-UNL-E. The results indicated that the optimal process conditions for S-UNL-E included mass ratios of phospholipid-cholesterol, phospholipid-breviscapine, phospholipid-sodium cholate, and phospholipid-stearamide were 2:1, 15:1, 7:1 and 7:1, respectively, and the mass of ethylenediamine tetramethylphosphonic acid (EDTMP) was 30 mg. The average particle size of S-UNL-E was 156.67 ± 1.76 nm, and Zeta potential was -28.77 ± 0.66 mv. S-UNL-E substantially increased the expression of ALP osteoblasts, elevated the content of osteocalcin protein and promoted the formation of mineralized nodules. Cells in the S-UNL-E group were densely distributed with integrated cell structure, and the actin filaments were clear and obvious. The findings demonstrated that S-UNL-E greatly promoted the differentiation and maturation of osteoblasts, and S-UNL-E (2.5 × 10⁸) produced the most favorable effect in differentiation promotion. In conclusion, the present study successfully constructed an S-UNL-E material characterized by high encapsulation and high stability, which could effectively promote osteogenic differentiation and bone formation.

Keywords: S-UNL-E; encapsulation; osteogenic differentiation; scutellarin.

Figure 1.

S-UNL-E particle size analysis and morphologic observation. (a) Results of particle size analysis. (b) S-UNL-E particle observation by transmission electron microscopy.

Figure 2.

Isolation and culture of osteoblasts. (a) Osteoblast morphology observed under white light. (b) Identification of osteoblast differentiation using ALP staining. (c) Determination of osteogenic differentiation by alizarin red staining. (d) Determination of cell proliferation activity by MTT. Scale bar: 100 μm.

Figure 3.

Determination of S-UNL-E regulation on the proliferation of osteoblasts by MTT. MTT assay was used to detect the effect of S-UNL-E on the proliferation of osteoblasts, and the proliferation curve was plotted based on the number of osteoblasts at the five time points of 24 h, 48 h, 72 h, 96 h and 120 h. (a-d) OD values treated at concentrations of 2.5 × 10⁶, 2.5 × 10⁷, 2.5 × 10⁸, and 2.5 × 10⁹. (e) Regulation of osteoblast proliferation by different concentrations ofS-UNL-E. (f) Differential regulation of S-UNL-E and S-UNL on osteoblast proliferation.

Figure 4.

The effect detection of S-UNL-E on osteoblast differentiation by ALP staining. (a) ALP staining result photographs. (b-e) ALP content determination, ALP content treated at concentrations of 2.5 × 10⁶, 2.5 × 10⁷, 2.5 × 10⁸, and 2.5 × 10⁹. F, The regulation comparison of ALP content in osteoblasts between S-UNL-E and S-UNL. Scale bar: 100 μm. ^##p < 0.01, compared with BC group.

Figure 5.

Alizarin red staining and osteocalcin content determination. (a) Alizarin red staining. (b-e) ALP content determination, results of osteocalcin content determination treated at concentrations of 2.5 × 10⁶, 2.5 × 10⁷, 2.5 × 10⁸, and 2.5 × 10⁹. (f) Regulation of osteocalcin content in osteoblasts between S-UNL-E and S-UNL. Scale bar: 100 μm. ^##p < 0.01, compared with BC group.

Figure 6.

Calcium nodules and actin detection. (a) Calcified nodules in each group were observed by scanning electron microscopy. (b) Cytoskeleton actin staining was observed by fluorescence microscopy. Scale bar: 50 μm.