Aluminium-based fluorinated counterion for enhanced encapsulation and emission of dyes in biodegradable polymer nanoparticles

Dye-loaded polymer nanoparticles, due to their high brightness and potential biodegradability, emerge as a powerful alternative to quantum dots in bioimaging applications. To minimize aggregation-caused quenching of the loaded dyes, we have recently proposed the use of cationic dyes with bulky hydrophobic counterions (also known as weakly coordinating anions), which serve as spacers preventing dye pi-stacking inside nanoparticles. However, so far this approach of counterion-enhanced emission inside polymer NPs has been limited to one fluorinated tetraphenylborate (tetrakis(pentafluorophenyl)borate, F5-TPB). Herein, we show that the counterion-enhanced emission approach is not limited to tetraphenylborates and can be extended to other types of anions, such as the aluminium-based anion, Al[OC(CF3)3]4− (F9-Al), which is much easier to scale up, compared to F5-TPB. It is found that F9-Al strongly improves the encapsulation efficiency of the octadecyl rhodamine B dye compared to the perchlorate counterion (97 ± 2 vs. 51 ± 2%), being slightly better than F5-TPB (92 ± 4%). Similarly to F5-TPB, F9-Al can effectively prevent aggregation-caused quenching of rhodamine inside NPs made of the biodegradable polymer, poly(lactide-co-glycolide) (PLGA), even at 50 mM dye loading. According to single-particle microscopy, the obtained NPs are 33-fold brighter than commercial quantum dots QD585 at 532 nm excitation and exhibit complete ON/OFF switching (blinking), as was originally observed for NPs based on F5-TPB. Importantly, NPs loaded with the rhodamine/F9-Al ion pair entered the cells by endocytosis, showing no signs of dye leaching, in contrast to rhodamine perchlorate, which exhibited severe leakage from NPs with characteristic accumulation inside mitochondria. Moreover, F9-Al surpassed the F5-TPB anion in stability of dye-loaded NPs against leaching, which can be attributed to the higher hydrophobicity of the former. Overall, this work shows that counterion-enhanced encapsulation and emission of cationic dyes inside polymer NPs is a general approach for the preparation of stable and highly fluorescent nanomaterials for bioimaging applications.

Reaction mixture was left for 2 hrs at 80 o C and then temperature was risen to 100 o C for one hour, afterwards the reaction was left at 50 o C over the weekend.
Afterwards, the reaction mixture was heated up to reflux and hot filtration was performed, separating greyish insoluble powder from toluene solution.Solution was let to cool down, put to freezer, and precipitated white crystals were filtered and washed with cold toluene and dried under vacuum, which yielded 5.23 g (5.37 mmol, 27 %) of Li[F9-Al].
Decreased product yields and weaker reactivity of LAH comparing to original protocol could be explained by insufficiently dry toluene and/or argon. 19
b This value probably corresponds to the amount of large aggregates of pure dye that cannot go through dialysis membrane.

Figure S6 .Figure S7 .
Figure S6.Effect of β-cyclodextrin (βCD) on absorption spectra of R18/ClO 4 in water.Approximately 2 µM solution of R18/ClO 4 dye in water was treated with different amounts of βcyclodextrin.Spectra demonstrate that β-cyclodextrin can help to dissolve aggregates of R18/ClO 4 dye in water, and the required concentration is about 1 mM.Dilution due to addition of βcyclodextrin was less than 10%.
Figure S9.Single-particle blinking.Different emission transients of 50 mM-loaded R18/F5-TPB (right) and R18/F9-Al (left) NPs under 0.6 W/cm 2 532 nm laser illumination.Recording speed was 33 frames per second.No significant difference in blinking behaviour was observed between the two types of NPs.

Table S2 .
Encapsulation efficiency of 50 mM dye-loaded NPs. a

Table S3 .
Quantum yield data of tested dye-loaded NPs. a a SDstandard deviation of the mean (n = 3).