25 mg (4.166 Â 10
À4 mmol) of elastin was dissolved in 125 μL of 0.04 M PBS
buffer (pH 9) and dispersed in 1.25 mL n-hexane containing 37.5 mg surfactant (3:1
weight ratio of Span 80 and Tween 80). Subsequently, the dispersion was
ultrasonicated under ice cooling for 60 s using a Branson sonifier W450 with a ¼
horn at a duty cycle of 30% and output control of 90%. Crosslinking was effected
by addition of PEG-DGE or BS3 followed by a further sonication for another 60 s.
The dispersion was further agitated for 45 min at room temperature before the
reaction was quenched by addition of 1.5 mL of acidic water (pH 3). Separation of
the microgels was achieved by centrifugation at 10,000 rpm for 30 min with
subsequent decantation of the supernatant. Microgels present in the aqueous layer
were carefully washed with hexane (2 Â 1.5 mL) and THF (4 Â 2.5 mL) in order
to remove the surfactants and unreacted elastin. The remaining organic solvents and
acid were removed by dialysis. Purified microgels were stored in Millipore water at
4
C for further use.
2.3 XTT Cytotoxicity Test
Cytocompatibility of the BS3-elastin microgels with molar ratio of 1:0.5 was
investigated by XTT-based cell proliferation assay (Roche XTT Cell Proliferation
Kit II, catalog no. 1465015) according to the manufacturer’s guidelines. The gels
were prepared on the bottom of 96-well polystyrene plates. Human umbilical vein
endothelial cells (HUVECs) (10,000 per well) and 500 μL of 10% fetal bovine
serum medium were added to the wells (1.77 cm
2 ), and the mixtures were incubated
in 5% CO 2 at 37
C for 96 h. To halt the experiment, aliquots of XTT stock solution
(5 mg/mL) were added to each experimental well group at a ratio of 50/100 μL/μL
of medium. Experimental plates were incubated for 4 h, and samples (100 μL) from
each well were transferred to 96-well plates and quantified using a TECAN reader
at a λ of 490 nm and a reference λ of 655 nm.
2.4 Sedimentation Analysis
Sedimentation of microgels was investigated with a LUMiFuge 114 separation
analyzer (L.U.M. GmbH, Germany). Measurements were performed in glass
tubes at acceleration velocities of 500–3,000 rpm at 20
C. The slope of the
sedimentation curves was used to calculate the sedimentation velocity, indicating
the colloidal stability of the samples.
Stimuli-Sensitive Microgels from Native Elastin: An Easy Approach for a Drug. . .
419
À4 mmol) of elastin was dissolved in 125 μL of 0.04 M PBS
buffer (pH 9) and dispersed in 1.25 mL n-hexane containing 37.5 mg surfactant (3:1
weight ratio of Span 80 and Tween 80). Subsequently, the dispersion was
ultrasonicated under ice cooling for 60 s using a Branson sonifier W450 with a ¼
horn at a duty cycle of 30% and output control of 90%. Crosslinking was effected
by addition of PEG-DGE or BS3 followed by a further sonication for another 60 s.
The dispersion was further agitated for 45 min at room temperature before the
reaction was quenched by addition of 1.5 mL of acidic water (pH 3). Separation of
the microgels was achieved by centrifugation at 10,000 rpm for 30 min with
subsequent decantation of the supernatant. Microgels present in the aqueous layer
were carefully washed with hexane (2 Â 1.5 mL) and THF (4 Â 2.5 mL) in order
to remove the surfactants and unreacted elastin. The remaining organic solvents and
acid were removed by dialysis. Purified microgels were stored in Millipore water at
4
C for further use.
2.3 XTT Cytotoxicity Test
Cytocompatibility of the BS3-elastin microgels with molar ratio of 1:0.5 was
investigated by XTT-based cell proliferation assay (Roche XTT Cell Proliferation
Kit II, catalog no. 1465015) according to the manufacturer’s guidelines. The gels
were prepared on the bottom of 96-well polystyrene plates. Human umbilical vein
endothelial cells (HUVECs) (10,000 per well) and 500 μL of 10% fetal bovine
serum medium were added to the wells (1.77 cm
2 ), and the mixtures were incubated
in 5% CO 2 at 37
C for 96 h. To halt the experiment, aliquots of XTT stock solution
(5 mg/mL) were added to each experimental well group at a ratio of 50/100 μL/μL
of medium. Experimental plates were incubated for 4 h, and samples (100 μL) from
each well were transferred to 96-well plates and quantified using a TECAN reader
at a λ of 490 nm and a reference λ of 655 nm.
2.4 Sedimentation Analysis
Sedimentation of microgels was investigated with a LUMiFuge 114 separation
analyzer (L.U.M. GmbH, Germany). Measurements were performed in glass
tubes at acceleration velocities of 500–3,000 rpm at 20
C. The slope of the
sedimentation curves was used to calculate the sedimentation velocity, indicating
the colloidal stability of the samples.
Stimuli-Sensitive Microgels from Native Elastin: An Easy Approach for a Drug. . .
419