This mixture was centrifuged at 12 000 rpm and at 4°C for 1 h. For Dox infusions, 500 lL of 3.68 × 10 -5 M Dox solution and 50 lL of original microgel sample were incorporated in 5 mL of PBS (pH 7.4) before centrifugation. The Dox loading level of the microgels used in this study was 31.5 % (mg Dox/mg polymer).
Bioconjugation of Transferrin to the Surface of Microgels:The conjugation of transferrin and albumin to the microgels was accomplished by using the same process. A 10 mg ml -1 stock solution of the proteins was made. 240 lL of this solution was then mixed with 300 lL of loaded gels in 0.01 M PBS at pH 7.4. Then, at least a 10-fold molar excess of 1-ethyl-3-(2-dimethylaminopropyl) carbodiimide hydrochloride was added to mediate the formation of an amide bond between carboxylic groups on the gel and amino groups on the protein.R6G-Loaded Microgel Assay: HeLa cells were grown on cover slips in 100 mm tissue culture dishes until 50 % confluency was reached. R6G-loaded hydrogel particles, conjugated to transferrin, were dispersed in high glucose Dulbecco's modified eagle medium (DMEM) supplemented with 10 % fetal bovine serum, 1 % penicillin, and 1 % amphotericin B. For the controls, non-conjugated R6G-loaded microgel particles, particles conjugated to bovine serum albumin, and particles in solution with, but not conjugated to transferrin, were used. Cells were incubated overnight in this system. After washing the coverslips with 10 mM PBS, the cells were fixed with 3-4 % paraformaldehyde, followed by three washes with PBS. The cells were examined under 20× magnification through differential interference contrast and epifluorescence. Quantitative analysis was based on the measurement of luminescence intensity per cell using ImagePro software.
Dox-Loaded Microgels Assay:One million HeLa cells were placed in each 60 mm tissue-culture dish and allowed to grow for two days. Dox-loaded hydrogel particles, conjugated to transferrin, were dispersed in high glucose DMEM, supplemented with 10 % fetal bovine serum, 1 % penicillin, and 1 % amphotericin B. HeLa cells were incubated for 36 h with Dox-loaded hydrogel particles conjugated to transferrin and control systems. The cells were then trypsinized, washed with PBS, and stained with Trypan Blue. [1][2][3] and stiffness [4][5][6] of carbon nanotubes are in excess of other fibrous materials and make them attractive as a structural material in future polymer composites. Critically, this reinforcement is effective only when the interfacial adhesion between the nanotube and surrounding polymer is substantial. We report in this work how the interfacial strength between a single carbon nanotube and a polymer matrix increases dramatically when the nanotube surface is chemically modified. This is shown by measuring the force required to pull individual nanotubes out of a polymer matrix using various embedded lengths. As opposed to our previous work, [7][8][9] the nanomechanical test is performed here in an electron microscope, which reveals a transition from nanotube pullout t...