Coumarin 6 is a small molecular fluorescent dye, which has been frequently used for the traceability of excipients in vitro. And here, it was used as a hydrophobic model probe to explore the formation of micelles indirectly.

Figure 1. The emission fluorescence intensity of coumarin 6 in water.

Coumarin in water

The emission fluorescent intensity of coumarin 6 in pure water, which was excited by the light with a maximum excitation wavelength at 441.7 nm, was exhibited in Figure 1. Negligible fluorescence was recorded by the spectrometer from 450 nm to 850nm, while only a small specific emission peak at wavelength of 883.6 nm was observed, which might be emitted by few parts of coumarin 6 molecules which were aggregated in some unique forms in water because of their extremely poor water- solubility.

Coumarin in acetone and micelles

As shown in Figure 2, a strong emission peak of coumarin 6 in acetone (20 μg/mL) at wavelength of 508.3 nm was recorded. The dramatic increase of emission fluorescent intensity might correspond to the increase of coumarin 6 solubility in organic solvent. What’s more, there was no emission peak detected at wavelength of 883.6 nm.

Figure 2. The emission fluorescence of coumarin 6 (20 μg/mL) in acetone and nanomicelles.

Fluorescence quenching

Figure 3 exhibited the emission fluorescence quenching process after a twice volume of pure water was added into 1 mL of acetone solution of coumarin 6 (10 μg/mL). After the addition of pure water into coumarin 6 acetone solution, the fluorescence intensity of coumarin 6 was detected immediately, which was decreased from some 450 to about only 50 within about 20 minutes. This might be caused by the hydrogen bound formed between water molecules and coumarin 6, just as previously reported on the quenching process between coumarin 102 and phenol molecules¹.

Figure 3. Fluorescence quenching process of coumarin 6 in acetone caused by the addition of water.

The solubility of coumarin in water was too low, the addition of water might also cause the decrease of emission fluoresce. Therefore the water added in the experiment was replaced by alcohol, the fluorescence quenching process could also be observed. Alcohol contained hydroxy groups, but the solubility of coumarin in alcohol was much higher that in water.

Nanomicelle formation

The emission fluorescence of coumarin 6 loaded nanomicelles were also detected. As shown in Figure 2, an emission peak at 492.3 nm was observed, which was much larger than that of coumarin 6 in pure water, indicating that the solubility of coumarin 6 in aqueous solution increased with the help of the amphiphilic copolymers. What’s more, this emission peak was shifted to a shorter wavelength (16 nm blue shift) in comparison with that of coumarin 6 in acetone. According to the previous study ², this emission band shift verified that the coumarin 6 molecules in the aqueous solution were located in an apolar microenvironment. The emission fluorescence of coumarin 6 loaded nanomicelles, which were stored at room temperature, could still be detected after one week with almost the same intensity. Besides, no emission fluorescence of the amphiphilic polymers was observed. These results demonstrated that the amphiphilic polymers in water can form some sealed shapes with hydrophobic inner parts, inside which coumarin 6 could be entrapped to prevent the fluorescence quenching caused by interaction with water molecules as well as to increase its solubility in water.

Figure 4. SEM image of the formed particles.

Actually, the concrete shapes of the sealed polymeric particles were characterised by SEM, which was exhibited in Figure 3.2.4_4. Some spherical shapes with a diameter size of about 100 nm were observed from the SEM image.

A little more thinking

Besides, a small specific emission peak at around 883 nm on the fluorescence spectrum of coumarin 6 loaded nanomicelles was observed in Figure 2, which was just as same as that of coumarin 6 dissolved in pure water. This indicated that not all of the coumarin 6 molecules were entrapped inside the polymeric sealed shapes, but parts in water. The emission fluorescent intensity of coumarin 6 loaded nanomicelles was much lower than that of coumarin 6 in acetone with the same concentration. This might be caused by the loss of coumarin 6 during the preparation of micellar suspensions and the interaction of coumarin 6 with water molecules.

In addition, according to the related literature³, for generating polymersomes in aqueous solution, polymers should possess a ratio of hydrophilic to total mass at around 35% ± 10%. Therefore, all the results above demonstrated that polymeric nanomicelles could be obtained using the synthesised amphiphilic block copolymers through the desolvation technique.