2D Covalent Organic Framework Direct Osteogenic Differentiation of Stem Cells

Abstract

2D covalent organic frameworks (COFs) are an emerging class of crystalline porous organic polymers with a wide-range of potential applications. However, poor processability, aqueous instability, and low water dispersibility greatly limit their practical biomedical implementation. Herein, a new class of hydrolytically stable 2D COFs for sustained delivery of drugs to direct stem cell fate is reported. Specifically, a boronate-based COF (COF-5) is stabilized using amphiphilic polymer Pluronic F127 (PLU) to produce COF-PLU nanoparticles with thickness of ≈25 nm and diameter ≈200 nm. These nanoparticles are internalized via clathrin-mediated endocytosis and have high cytocompatibility (half-inhibitory concentration ≈1 mg mL^-1 ). Interestingly, the 2D COFs induce osteogenic differentiation in human mesenchymal stem cells, which is unique. In addition, an osteogenic agent-dexamethasone-is able to be loaded within the porous structure of COFs for sustained delivery which further enhances the osteoinductive ability. These results demonstrate for the first time the fabrication of hydrolytically stable 2D COFs for sustained delivery of dexamethasone and demonstrate its osteoinductive characteristics.

Keywords: 2D nanoparticles; covalent organic frameworks (COFs); drug delivery; tissue engineering.

Figure 1.

Schematic showing fabrication of hydrolytically-stable COF-PLU nanomaterials for sustained delivery of therapeutics (Dex – dexamethasone) for directing stem cell differentiation.

Figure 2.. Synthesis and characterization of 2D COF-PLU nanoparticle.

(A) Schematic showing solvothermal condensation of HHTP and BDBA producing colloidal COF-5 which are unstable in physiological condition. Wrapping of COF-5 with an amphiphilic polymer (pluronic F127 (PLU)) produces a stable COF-PLU nanoparticles. (B) Hydrolytic stability of COF-5 indicating its rapid degradation in water. Inset show complexation of colloidal COF-5 with an amphiphilic polymer pluronic F127 (PLU) forms self-assembled nanoparticles (COF-5 + PLU) which precipitated out. (C) Dynamic light scattering profiles (blue histogram) and transmission electron micrographs (inset) show size of COF-PLU nanoparticles. (D) Atomic force micrographs of COF-PLU reveal the thickness to be ~ 20–25 nm. E) Transmission electron micrographs of COF-PLU nanoparticles at day 1, 3 and 7 indicating their stability in water. (F) ATR-FTIR profile of COF-PLU reveals presence of characteristic peaks for both COF-5 (1347 & 1332) and PLU (1100) in COF-PLU. G) Thermogravimetric analysis profile of COF-PLU revealing presence of two consecutive weight loss events at ~400 °C (characteristics of PLU) and ~650 °C (characteristics of COF-5). The differential temperature is then plotted against time and is shown as differential thermogravimetric (DTG) analysis.

Figure 3.

Cellular interaction of COF-PLU. (A) Cytocompatibility of COF-PLU is evaluated by subjecting hMSc with varying concentration of COF-PLU nanoparticles. Half-maximal inhibitory concentration (IC₅₀) was determined by concentration of COF-PLU nanoparticles to inhibit cell viability by half. (B) Cell cycle analysis of hMSCs treated with COF-PLU nanoparticles (15, 200 and 300 μg/mL) indicating no significant changes in cell cycle upon treatment with COF-PLU nanoparticles. (C) Fluorescence micrographs of hMSC treated with FITC-tagged COF-PLU nanoparticles indicate their cellular internalization. (D) Schematic for proposed cellular internalization pathway of COF-PLU nanoparticles. (e) Flow cytometry profiles of FITC-tagged COF-PLU nanoparticles confirming clathrin-mediated endocytosis as major cellular internalization pathway of COF-PLU (n = 3 in each case).

Figure 4.

Synthesis and characterization of therapeutic loaded COF-PLU nanoparticles. (A) Transmission electron micrograph of dexamethasone (Dex) loaded COF-PLU (COF-PLU/Dex). (B) FTIR profile of COF-PLU, dexamethasone, dexamethasone loaded COF-PLU nanoparticles (COF-PLU/Dex). (C) Entrapment efficiency as well as loading capacity of dexamethasone on COF-PLU nanoparticles. (D) Release profile dexamethasone from COF-PLU/Dex at different pH to mimic extracellular (pH ~7.4) and intracellular microenvironment (pH ~5 (endosome/lysosome)). (E) COF-PLU nanoparticles induce osteogenic differentiation of hMSCs and sustained delivery of dexamethasone (Dex) from Dex loaded COF-PLU (COF-PLU/Dex) further enhance the osteogenic differentiation. hMSCs are cultured in osteoconductive (OC) media and treated (n = 3) with COF-PLU, Dex (exogenous), and COF-PLU/Dex. hMSCs in OC media is used as control. ALP expression (staining) and activity is measured at Day 7 and 14 using BCIP/NBT staining and ALP kinetic assay. COF-PLU/Dex show strong ALP expression and activity on both day 7 and 14. (F) Treatment with COF-PLU/Dex enhances the expression of RunX2, a key osteogenic protein as indicated by western blot analysis. (G) Significanlty higher production of mineralized matrix is observed in hMSCs treated with COF-PLU/Dex compared to control or dexamethasone treated hMSCs. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, and ****P-value < 0.0001.

Figure 5.

Mechanistic insight of osteogenic activity of COF-PLU nanoparticles. (A) Relative ALP activity of hMSC treated with COF-PLU, COF-5, and PLU at day 7. hMSCs treated with and without dexamethasone is used as positive and negative controls respectively (n = 3 in each group, **P-value < 0.01, ***P-value < 0.001). (B) To investigate the mechanism of osteoinductive characteristics of COF-PLU, hMSCs are treated with inhibitors of major osteogenic pathways (Wnt, TGF-β, JNK, p-38, and ERK) in presence and absence of COF-PLU. ALP activity of hMSCs was determined at day 4, 8, and 12 (n = 3 in each group, **P-value < 0.01, ***P-value < 0.001). (C) Schematic showing ability of COF-PLU trigger JNK pathway leading to osteogenic differentiation via upregulation of RunX2.