Preparation and in vitro evaluation of novel cross-linked chondroitin sulphate nanoparticles by aluminium ions for encapsulation of green tea flavonoids

Abstract

Chondroitin sulphate is a sulphated glycosaminoglycan biopolymer composed over 100 individual sugars. Chondroitin sulphate nanoparticles (NPs) loaded with catechin were prepared by an ionic gelation method using AlCl₃ and optimised for polymer and cross-linking agent concentration, curing time and stirring speed. Zeta potential, particle size, loading efficiency, and release efficiency over 24 h (RE₂₄%) were evaluated. The surface morphology of NPs was investigated by scanning electron microscopy and their thermal behaviour by differential scanning calorimetric. Antioxidant effect of NPs was determined by chelating activity of iron ions. The cell viability of mesenchymal stem cells was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide assay and the calcification of osteoblasts was studied by Alizarin red staining. The optimised NPs showed particle size of 176 nm, zeta potential of -20.8 mV, loading efficiency of 93.3% and RE₂₄% of 80.6%. The chatechin loaded chondroitin sulphate NPs showed 70-fold more antioxidant activity, 3-fold proliferation effect and higher calcium precipitation in osteoblasts than free catechin.

Fig. 1

Contribution percent of the studied variables on the particle size, zeta potential, loading efficiency and release efficiency of catechin from chondroitin NPs

Fig. 2

Catechin release profiles from (a) different studied chondroitin sulphate nanoparticles in pH 7.4, (b) the optimised nanoparticles in two different pH

Fig. 3

SEM micrograph of the optimised catechin loaded chondroitin sulphate NPs of S₉₅₀₀ C_0.5 A_0.5 T₂₅

Fig. 4

DSC thermograms of (a) catechin loaded chondroitin sulphate nanoparticles of S₉₅₀₀ C_0.5 A_0.5 T₂₅, (b) free catechin, (c) chondroitin sulphate, (d) Poloxamer 188

Fig. 5

Antioxidant activity of free catechin and optimised catechin loaded chondroitin sulphate NPs in chelating Fe²⁺ ions at different time intervals

Fig. 6

Proliferation of mesenchymal stem cells after incubation with different concentrations of free catechin or catechin nanoparticles by MTT assay after (a) three days, (b) seven days

Fig. 7

Alizarin red staining of mesenchymal stem cells cultured with various concentrations of free catechin, catechin loaded NPs and chondroitin sulphate NPs (without catechin) in 14 and 21 days of exposure