To evaluate the potential of a cationic calixarene-based nanoaggregate as a novel nanocarrier for curcumin ocular delivery by investigation of anti-inflammatory effects. Methods: An amphiphilic calix[4]arene derivative bearing four choline groups was assembled with curcumin to give a clear colloidal solution in PBS medium. Size and surface charge of the nanostructured system were derived from DLS analysis and zeta potential measurements. UV-vis spectroscopy and HPLC analyses were performed to study the curcumin solubility and chemical stability alone and loaded into the calix[4]arene nanoaggregate. Corneal cells cultures were treated with different concentrations of curcumin, calixarene alone and curcumin-loaded calixarene, at 24hrs MTT assay was performed. Murine macrophages cells (J774) pre-treated for 2hrs with different concentrations of curcumin, calixarene alone and curcumin-loaded calixarene and stimulated for 24hrs with LPS (10?g/ml), were used to test the anti-inflammatory effects of compounds. At 24 hrs MTT assay and western blot analysis were performed. An experimental uveitis was induced in rats by injection of 200 ?g of LPS into the foot; curcumin, calixarene alone and calixarene loaded were administered as pre-treatment for 3 days before and 7 days after the induction of uveitis. Animals at 16 and 72 hrs were sacrificed to perform histology and immunohistochemical staining. Results: The curcumin-loaded calixarene showed a mean hydrodynamic diameter of 82 nm (Z average), polydispersity index of 0.2 and zeta potential of 23 mV. The drug loading capacity was calculated to be about 10%. Calixarene enhances curcumin solubility (90 µg/mL per 1 mg/mL of carrier) and its chemical stability in PBS at pH 7.4. No cytotoxicity was reported using curcumin alone (range 0.05-0.00125) and calixarene with or without curcumin (range 0,25-0,015 mg/ml). The anti-inflammatory activity of curcumin loaded into calix[4]arene was showed through the modulation of of NF-?B activation and reducing I?B? degradation (p<0.001 vs LPS control), reducing COX-2 and iNOS expression and the nitrite levels (p<0.001 vs LPS control). In vivo studies confirmed the anti-inflammatory effects of curcumin and particularly of curcumin-loaded calixarene (p<0.001). Conclusions: An amphiphilic calix[4]arene derivative that spontaneously self-assembles in discrete cationic nanoaggregates was designed to optimize the ocular delivery of curcumin. The capability of the calixarene-based nanoaggregate to load and release curcumin was demonstrated. Using murine macrophage J774 cells LPS-stimulated and an experimental uveitis model in the rat the enhanced anti-inflammatory efficacy of curcumin was proved, showing that the calixarene nanoaggregate is a promising novel nanocarrier for ocular delivery of curcumin.
Calixarene-based nanoformulation for ocular delivery of curcumin: in vitro and in vivo anti-inflammatory activity evaluation
G Granata;
2016
Abstract
To evaluate the potential of a cationic calixarene-based nanoaggregate as a novel nanocarrier for curcumin ocular delivery by investigation of anti-inflammatory effects. Methods: An amphiphilic calix[4]arene derivative bearing four choline groups was assembled with curcumin to give a clear colloidal solution in PBS medium. Size and surface charge of the nanostructured system were derived from DLS analysis and zeta potential measurements. UV-vis spectroscopy and HPLC analyses were performed to study the curcumin solubility and chemical stability alone and loaded into the calix[4]arene nanoaggregate. Corneal cells cultures were treated with different concentrations of curcumin, calixarene alone and curcumin-loaded calixarene, at 24hrs MTT assay was performed. Murine macrophages cells (J774) pre-treated for 2hrs with different concentrations of curcumin, calixarene alone and curcumin-loaded calixarene and stimulated for 24hrs with LPS (10?g/ml), were used to test the anti-inflammatory effects of compounds. At 24 hrs MTT assay and western blot analysis were performed. An experimental uveitis was induced in rats by injection of 200 ?g of LPS into the foot; curcumin, calixarene alone and calixarene loaded were administered as pre-treatment for 3 days before and 7 days after the induction of uveitis. Animals at 16 and 72 hrs were sacrificed to perform histology and immunohistochemical staining. Results: The curcumin-loaded calixarene showed a mean hydrodynamic diameter of 82 nm (Z average), polydispersity index of 0.2 and zeta potential of 23 mV. The drug loading capacity was calculated to be about 10%. Calixarene enhances curcumin solubility (90 µg/mL per 1 mg/mL of carrier) and its chemical stability in PBS at pH 7.4. No cytotoxicity was reported using curcumin alone (range 0.05-0.00125) and calixarene with or without curcumin (range 0,25-0,015 mg/ml). The anti-inflammatory activity of curcumin loaded into calix[4]arene was showed through the modulation of of NF-?B activation and reducing I?B? degradation (p<0.001 vs LPS control), reducing COX-2 and iNOS expression and the nitrite levels (p<0.001 vs LPS control). In vivo studies confirmed the anti-inflammatory effects of curcumin and particularly of curcumin-loaded calixarene (p<0.001). Conclusions: An amphiphilic calix[4]arene derivative that spontaneously self-assembles in discrete cationic nanoaggregates was designed to optimize the ocular delivery of curcumin. The capability of the calixarene-based nanoaggregate to load and release curcumin was demonstrated. Using murine macrophage J774 cells LPS-stimulated and an experimental uveitis model in the rat the enhanced anti-inflammatory efficacy of curcumin was proved, showing that the calixarene nanoaggregate is a promising novel nanocarrier for ocular delivery of curcumin.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
