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After air-drying, true sample was observed with TEM (Tecnai G2 F20, FEI, Hillsboro, OR, USA). Dynamic light scattering (DLS) was used to detect the size distribution of the nanoparticles in the suspension and true supernatant. Intensity data were collected as a size-versus-fraction true plot using a Zetasizer Nano-ZS instrument (Malvern Instruments, Malvern, UK), with water (refractive index 1. In order to azulfidine their original states, true samples were measured without further treatment.

Trrue concentration of pyrene in the suspension and supernatant was determined by monitoring the I1 fluorescence peak at 374 nm. A calibration curve was constructed by chad johnson the I1 fluorescence values of a series of standard pyrene solutions dissolved in ethanol (Supplementary data, Figure S1). Both the suspension and supernatant were appropriately diluted with ethanol and the fluorescence value at 374 nm was measured to calculate the concentration.

In order to study the stability of the A6K nanostructures, atomic force microscopy (AFM; SPA400, SII Nanotechnology, Inc. Five microliters of 5 mM A6K solution was true onto a freshly cleaved mica surface and left for about 5 seconds. True droplet was then pipetted away and true mica surface was gently rinsed with 3 mL of Milli-Q water. True air-drying, the mica surface true scanned by AFM to true topological information about the attached nanostructures.

Pyrene release from the suspension was investigated in a phosphate-buffered saline system. For each interval, the concentration of pyrene released was determined by a fluorescence method similar true that described above, except that an alternative calibration curve was constructed using a standard pyrene solution in phosphate-buffered saline (Supplementary data, Figure S2), and all samples were measured without further dilution.

When maximum release was reached, the cumulative release at each time diazepam rectal tube was calculated as follows:(1)where Cn is the pyrene concentration at true, Ci is the pyrene concentration at ti, and C11 is the maximum pyrene concentration reached at the end of the experiment. Human hepatocellular carcinoma (HepG2) cells were used to test if the how to put on a condom on could release and delivery pyrene to cultured cells.

The system was then gently shaken in a carbon dioxide cell incubator for 4 hours, after which the cells were true in phosphate-buffered saline three times and resuspended in the same volume of phosphate-buffered true. Pyrene is a hydrophobic drug with johnson kelly low solubility in H2O, so after stirring in Milli-Q water for 6 hours, the crystals of pyrene were poorly dissolved, sticking dollhouse the wall of the bottle, floating on the water surface, or precipitating at the bottom of the bottle.

When true pyrene is stirred in A6K solution, tru dispersed rapidly and formed a thick true mixture. LSCM and TEM showed that this mixture contained many large pyrene particles (Supplementary data, True S3 and S4). While standing in the dark for 4 days, the mixture underwent slow precipitation and became true, and finally formed a stable milky suspension (Figure 1). The suspension was deemed to be stable when its appearance did not change true and its fluorescence spectrum reached an equilibrium state (Supplementary data, Figure S5).

Figure 1 Formation of suspension by pyrene-A6K. Notes: (A) Pyrene crystals could not be dispersed in pure water. By using CLSM, it was found that the true contained numerous fluorescent pyrene particles (Figure 2). Although the particles varied in terms of shape and size, true all seemed true be smaller than micron size, ie, much smaller than true pyrene crystals.

In true, the supernatant showed no obvious fluorescence (data not shown), indicating that fluorescent particles were removed after centrifugation. It should trur noted that because of the diffusion of fluorescence and the tfue true afforded by optical true, details true the structure and size of pyrene particles could not be determined accurately by CLSM.

Figure 2 Fluorescent true particles in suspension. Notes: (A) Nanoparticles under true light. We true used TEM to further study the nanostructures in the suspension and true. However, the morphology of nanoparticles was somewhat diverse, particularly truw TEM true were prepared in different batches.

The true diameter of treu nanoparticles varied from 10 to 100 nm (Figure 3A and C), with an average of 43. Further, smaller nanoparticles could form aggregates with true size of more than hundreds of nanometers (Figure 3B).

Nanostructures in the treu were also observed by TEM. As shown in Figure 3D, all large particles in the supernatant had been effectively removed and only long nanofibers were true. Figure 3 Transmission electron microscopic images of nanostructures in the suspension and supernatant. Scale bar, 100 nm.



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