Degradation studies under 510 nm irradiation

Although all gels were extensively washed with acetone and water, Gel 1 still showed a slight colouring of the solvent. This was maybe due to the large BODIPY content used in the synthesis of this hydrogel. Small amounts of unreacted compound 4 (Diol-BODIPY) are probably still leaching out of the polymer matrix.

However, since already only a miniscule amount of BODIPY is enough for a strong response in fluorescence and free BODIPY should have no significant influence on the gel degradation, no further washings were performed.

The irradiation process was carried out in acetone at approximately 5 °C over the course of 5 days. Not only did the gels decrease in size but they also changed in colour and consistency. A comparison of the gels before and after irradiation, photographed under UV-light can be seen in Figure 40.

61 Figure 40: Comparison of BODIPY-hydrogels with different fluorophore concentration before and

after irradiation at λ= 510 nm for 5 days, photographed under UV-light. Left Gel 1 (1:2), middle Gel 2 (1:28), right Gel 3 (1:140).

Gel 2 and Gel 3 lost most of the glow under UV- light, though not visible in Figure 40 a faint glow still remained. The Hydrogels visibly shrunk in size and partly lost their structural integrity, which later manifested in disintegration of the gels upon heavy shaking. This indicates breakage of some crosslinks, however, not enough to allow a full dissolution of the gel.

Gel 1 shows like Gel 2 and Gel 3 a slight decrease in size, even though less than for the other two. In contrast to the other two, an interesting change in colour can be seen upon placing it under UV-light. The colour changed from yellow to orange.

This leads to the assumption that the BODIPY- framework was not fully destroyed during the photocleavage process. The colour shift from yellow to orange rather indicates a further advanced leaching-out-process of the BODIPY out of the hydrogel into the solvent, as this effect was also observed during the handling with various BODIPY solutions of different concentration.

before

after 1 cm

1 cm

62 Most probably the observed higher rate of degradation of Gel 2 and Gel 3 compared to Gel 1 is due to aggregation caused quenching. The high concentration of BODIPY leads to a close vicinity of the photocages resulting in a quenching effect. The lower concentration in Gel 2 and Gel 3 allowed an increased distance of the BODIPY molecules which prevented an interaction between them.

Additionally, a higher BODIPY concentration can lead to a strong absorption of the irradiated light, in turn preventing a deeper penetration of the radiation into the hydrogel since the light is already absorbed at the edges of the hydrogel. The photocleavage effect can therefor only take place in the outer layers of the hydrogel. In general, in order to achieve an efficient degradation process, it is not only required to use the right concentration but also ensure a uniform distribution within the polymer with no local accumulations. Finally, the differences before and after the irradiation were also visible under ambient light, depicted in Figure 41.

The colour of Gel 2 and Gel 3 clearly faded, the solution of gel 1 however, intensified a bit in colour. This may be due to some unreacted and not incorporated BODIPY still leaching out of the hydrogel over the duration of 5 days.

Figure 41: Comparison of BODIPY-hydrogels with different fluorophore concentration before and after λ= 510 nm irradiation after 5 days. Left Gel 1 (1:2), middle Gel 2 (1:28), right Gel 3 (1:140).

before

after 1 cm

1 cm

63 4.10.2 Degradation studies under 365 nm irradiation

The irradiation process with light at λ= 365 nm was carried out under the same conditions as for λ= 510 nm. However, already after 72 h a significant change could be observed.

In Figure 42 and Figure 43 a comparison of the gels before and after irradiation can be seen under ambient and UV-light, respectively. Gel 2 and Gel 3 completely dissolved and a colourless liquid remained and Gel 1 significantly shrunk in size, the slight colouring of the solvent completely faded.

Figure 42: Comparison of BODIPY-hydrogels with different fluorophore concentration before and after irradiation with light at λ= 365 nm after 72 h. Left Gel 1 (1:2), middle Gel 2 (1:28), right Gel 3

(1:140).

Analogous to the irradiation at 510 nm Gel 1 degraded at the slowest rate. As described above this may be caused by a self-quenching effect, limiting the practicable amount of incorporated BODIPY.

before

after 1 cm

1 cm

64 Based on the information received from the experiments, the optimum concentration for an effective degradation should therefore be in the area between Diol:Jeffamine = 1:28 and Diol:Jeffamine = 1:140.

Figure 43: Comparison of BODIPY-hydrogels with different fluorophore concentration before and after irradiation with λ= 365 nm light after 72 h. Left Gel 1 (1:2), middle Gel 2 (1:28), right Gel 3

(1:140).

In regard to the degradation rate of Gel 2 and Gel 3 the setup was checked after 24 h to ensure a working experiment. From a subjective point of view the degradation of the Gel 2 was slightly faster than that of Gel 3.

Finally, these experiments showed that only very small BODIPY concentrations have to be implemented into a polymeric structure in order to achieve photosensitive degradation behaviour. Too high concentrations can have an advert effect and limit the degradation rate. This indicates the possibility to synthesise photolabile polymers with only marginal effect on polarity or other properties as only small amounts of fluorophore are introduced in the polymeric structure.

before

after 1 cm

1 cm

65 4.10.3 Degradation studies of a control gel

As a control, a hydrogel has been prepared without any addition of BODIPY compounds. The simple, crosslinked polyurea gel has been synthesised analogous to the BODIPY hydrogels and was irradiated with λ= 365 nm light for 5 days. In Figure 44 a comparison of the hydrogel before and after irradiation with λ= 365 nm can be seen. No degradation could be observed, demonstrating a stable polymeric backbone during UV- irradiation.

Figure 44: Comparison of a hydrogel synthesised analogous to BODIPY-hydrogels but without fluorophore addition before and after irradiation with light at λ= 365 nm for 5 days.

The reason why the gels degraded much faster under λ= 365 nm irradiation than under λ= 510 nm despite a higher absorbance of the fluorophore at λ= 510 nm may be due to different power outputs of the used lamps. The actual brightness of the lamps could not be determined, however, the power output of the UV-lamps is roughly tenfold higher than that of the used green light LEDs with 64 W compared to the 6 W, respectively. Additionally, the light spectrum of the UV-lamp is much broader and less well defined as the light from the LED. Overall, giving an explanation for the apparently higher degradation rate at λ= 365 nm compared to λ= 510 nm.

before

after 1 cm

1 cm

66 4.10.4 Kinetic studies of the hydrogel degradation

Since the different power outputs of the used lamps has been a major parameter affecting the degradation rate in the previous experiments, the process was further examined. To this end the photo degradation of the hydrogels was observed via fluorescence emission spectroscopy on a Jobin Yvon Fluorolog 3 spectrometer allowing irradiation at the different wavelengths with roughly the same intensity as well as recording the absorbance and emission spectra after regular time intervals.

Gel 3 was used for the experiment. It was stored in deionized water and used in its swelled state, the sample was placed between 2 glass slides without any additional solvent.

In Figure 45 the fluorescence emission spectra recorded after regular time intervals can be seen. The gel was continuously excited with light at λ= 365 nm.

67 Figure 45: Fluorescence emission spectra of gel 3 recorded after regular time intervals and continuous irradiation at λ= 365 nm. Top (full spectrum), bottom (zoomed).

A continuous degradation process can be observed with a reduced emission intensity of 91 % at the maximum after 180 min.

Analogous, the gel was irradiated at 510 nm as well. In Figure 46 the fluorescence emission recorded after regular time intervals and continuous irradiation at λ= 510 nm is depicted.

In contrast to the irradiation experiments in the solvent it can be seen that the degradation process is considerably faster at λ= 510 nm compared to the irradiation at λ= 365 nm. After 180 min the fluorescence intensity at the emission maximum was reduced to 76 %. Furthermore, the kinetics at the different wavelengths are found to be in correlation with their absorbance intensities.

500 525 550 575 600 625 650 675

0

520 530 540 550 560 570 580 590

500000

68 Irradiation near the absorption maximum leads to the fastest degradation rate, at λ= 510 nm the hydrogels degrade roughly the factor 2.5 faster than at λ= 365 nm.

This is most likely due to the higher absorbance at 510 nm which is also by a factor of 2.5 higher compared to at 365 nm.

Figure 46: Fluorescence emission spectra of Gel 3 recorded after regular time intervals and continuous irradiation at λ= 510 nm light, Top (full spectrum), bottom (zoomed).

Overall, in combination with the results of the degradation experiments in the solvent in chapter 4.9.1, as well as of the control gel in chapter 4.10.3 it can be proven that the degradation process observed in Figure 41 as well as Figure 42 is in fact due to photocleavage of the BODIPY-compound. Additionally, it can also be seen that the absorption and emission of the incorporated BODIPY does not significantly change compared to its free unbound state. The maximum absorbance and emission for the unbound compound 4 (Diol-BODIPY) were

550 575 600 625 650 675

0

540 550 560 570 580 590

1200000

69 measured at λ= 538 nm and λ= 557 nm respectively and at λ= 526 nm and λ= 547 nm for the incorporated compound.

The small difference was expected due to the incorporation in the macromolecular system and possible changes in the chemical environment.

70

5. Outlook and conclusion

In conclusion, the work presented here reports the synthesis of a novel bifunctional green-light photo-labile BODIPY monomer, compound 4 (Diol-BODIPY), and its incorporation in different macromolecular systems. Compound 4 was successfully derivatised from literature compound 1 (BODIPY). Different synthetic approaches were tried with initially trying to first hydroxylate and then hydrolyse the structure, finally inverting the order of operations, the hydrolysis step at first and then the hydroxylation, allowing for better yields. An interesting finding was that the hydrolysis conditions had to be varied depending on the presence of a hydroxide functionality in position 3 in order to achieve a successful synthesis. Since the product had to be purified after each step via dry column vacuum chromatography, decreasing the yield significantly, a one-pot synthesis was tried, however led to no isolatable product. The final successful synthesis of the novel BODIPY compound was proven using ¹H-NMR, HSCQ, MS and UV-Vis characterisation. Furthermore, the reaction intermediates were characterized as well.

The Diol-BODIPY was then incorporated into a polymeric backbone in different concentrations, namely, BODIPY: Jeffamine 1:1, 1:3 and 1:15, and their photo-responsive degradation behaviour was investigated. Initially, the degradation studies were initially carried in DMF as solvent, however it was found that the BODIPY-oligomers degrade in DMF in the dark as well. The solvent was therefor changed to MeOH, where good stability in the dark was observed. From the irradiation studies it could be seen that both with λ= 365 nm irradiation as well with λ= 510 nm a photocleavage was achieved. Although the degradation process first appeared to be faster at 365 nm irradiation than at 510 nm, this was more likely based on the different power output of the lamps used at the certain wavelengths.

This proposed reason was further verified by the irradiation of synthesized hydrogels in later experiments. Despite the usage of the polar Jeffamine ED-2003 as co-monomer, no water-soluble oligomers could be produced. The exact reason for the insolubility in water could not be determined. Further research in this area may lead improvements in solubility and possible uses in biomedicine as carrier polymers for different pharmaceuticals with stimuli responsive release.

71 Finally, crosslinked hydrogels were synthesised with varying ratios, (Diol-BODIPY) : Jeffamine-ED-2003 = 1:2, 1:28 and 1:140, and their degradation behaviour was analysed. The different hydrogels were first irradiated in solution in acetone with light at λ= 365 nm and λ= 510 nm for 3 days. Therefore, the gels were placed in acetone in order to allow any degradation products to leach out and to prevent the gels from drying out. It could be seen that the hydrogels degrade upon irradiation, which served as a proof of principle. To further establish the kinetics of the degradation process fluorescence emission spectra of the hydrogels were recorded after regular time intervals and continuous irradiation at specific wavelengths. As mentioned above, in contrast to the preliminary experiments in solution the degradation was found to be wavelength dependent as expected. The experiments showed that the photocleavage was about 2.5 times faster upon irradiation with light at λ= 510 nm compared to at λ= 365 nm, correlating with the absorbance intensities at the respective wavelength.

Overall, from the experimental data presented herein it could be seen that the concentration of BODIPY has a significant influence on the degradation process.

At higher concentrations a decrease in the degradation rate could be observed, which can be most probably attributed to quenching effects. Additional problems can arise due to an uneven distribution of BODIPY within the polymer. However due to its limitations in synthesis it is very hard to control the exact polymeric sequence. In order to make this process more efficient an optimal concentration of incorporated BODIPY would have to be determined. From the results of this work it seems like the optimal concentration lies in the broad range of 1:28 and 1:140 (Diol-BODIPY: Jeffamine ED-2003). However, by a change of the co-monomeric material this ratio can change depending on its molecular mass. Additionally, structural changes on the bifunctional Diol-BODIPY compound could be carried out to adjust the kinetics.

BODIPY polymers are a very promising attempt in the development of stimuli responsive polymers acting as carrier matrices for pharmaceuticals. The green-light induced photocleavage could release drugs in a controlled manner. The mild visible light causes no tissue damage and the degradation products of Jeffamine as well as BODIPY are widely regarded as harmless.

72 These newly developed polymers and their inherent visible light stimulus open new ways in bio-medicinal therapies and could drastically improve a fast and non-invasive release of active compounds in high local concentrations.

73

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