Abstract
The aim of this research was to characterize and evaluate the innovative effects of the Thai herbal extract, Andrographolide (AG), on the human suprapatellar fat pad (SPFP) in the osteoarthritis (OA) patients and also develop and optimize the formulation of AG using Poly Lactic-co-Glycolic Acid (PLGA) nanocarriers and gelatin-based hydrogel. Suprapatellar fat pad adipose-derived stem cells (SPFP-ASCs) were isolated by enzymatic digestion, cultured and analyzed by light microscopy to observe morphology and growtAh. The cells treated with AG or without AG were characterized in vitro by immunophenotype, gene expression, and ability to differentiate into multilineage potential to be further differentiated into the osteogenic, adipogenic and chondrogenic cells under the appropriate inductions. The medium SPFP-MSCs cells showed uniform morphology and good proliferation. They expressed CD29, CD73, and CD105 but were negative for CD34 and CD45. Multilineage potential became positive for Alizarin Red S, Oil Red O, Toluidine Blue, and Alcian Blue staining while the effect of AG revealed that AG had no cytotoxic effects when the concentration was less than 12.5 μg/mL. Interestingly, AG had significantly enhanced osteogenesis and chondrogenesis as evidenced by a the significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, AG can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runt-related transcription factor 2 (Runx2), Osteopontin (OPN), SRYBox Transcription Factor 9 (Sox9), and Aggrecan. In contrast, AG suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and the expression levels for adipogenic-specific genes for Peroxisome Proliferator-Activated Receptor gamma 2 (PPAR-γ2) and Lipoprotein lipase (LPL). For these therapeutic properties, AG was successfully prepared the nanoparticles by the single emulsion solvent evaporation method and incorporated with gelatin-based hydrogel. The results of in vitro release patterns showed that the AG significantly delayed the AG release more than 1 month when combined with gelatinbased hydrogel and successfully displayed a long term sustained release almost 2 months for implantation and over 2 months for injection providing a novel strategy for the local management of osteoarthritis disease. Therefore, AG has great potential as the therapeutic agent derived from natural sources for regenerative medicine. Furthermore, this research aims to develop a pharmacokinetic model of an intra-articular drug delivery system for predicting drug concentrations targeted to mesenchyme stem cells. Using ADMET PredictorTM program, the physical and chemical properties of AG were predicted from the chemical structure. Then we develop and test a model of the formulation when injected into the knee joint. By using GastroPlus® 9.8.2 program and using the formulation as 0.1, 1, 5, and 10 mg solution, the amount of AG was predicted at the synovial fluid, plasma, cartilage, intimal, subintimal and lymph node. The amount of AG in different layers is the dosage of the AG that can be completely dissolved since the drug is injected into the knee joint as a clear liquid formulation. And from the prediction, it was found that there was no precipitation. The elimination of AG in rats, normal humans, and humans with rheumatoid arthritis is a first-order reaction. The elimination of AG in rats is the slowest when comparing with that in normal humans and humans with rheumatoid arthritis. In normal humans, AG can be eliminated better than in humans with rheumatoid arthritis so elimination time of AG in normal humans is shorter than humans with rheumatoid arthritis. Moreover, it was predicted that approximate 30% of the AG in the synovial fluid is in free form and the AG was not able to enter the lymph node. Since this study has not yet developed a model of AG as a delivery system that may also be able to enter the lymph node. In addition, for delivery system, AG can be specifically targeted to the MSCs located at the knee joint. Unlike conventional AG small molecule drugs, the pharmacokinetics of AG delivery system can be simulated using the theory of Target-mediated drug disposition (TMDD) model. We have successfully developed this model using Berkeley Madonna program as well in this study.
