S 450

Oxidized zeta potential measurements were performed in a Brookhaven potential analyzer (Brookhaven Instruments Corp., Holtsville, NY, USA). Ultrasonication of suspensions of pristine MWCNT, MWCNT-Ox, MWCNT-IA, and MWCNT-MMI was performed before both determinations. MWCNTs suspensions were prepared in 1 mM KCl solution at different pH (4.0, 7.0, and 9.0) and then sonicated. Sonication and ultrasonication of MWCNT samples were performed with an ultrasonic cleaning bath Power Sonic 410 and Branson S-450 digital sonifier equipped with a microtip, respectively. Different sonication cycles (up to three) were performed for all MWCNTs. Cycles 1 and 2 consisted in 2 min of sonication, then 1 min of ultrasonication was applied to MWCNT through a tapered microtip. In the third cycle, 10 min of ultrasonication was applied. For all ultrasonication, the applied amplitude was 40%. For zeta potential and particle size determinations of MWCNTs, 10 μL of MWCNT stock suspensions was taken with a Gilson Pipetman Classic 10 μL (P10) and mixed with 10 mL of bifiltered 1 mM KCl at pH 4.0, 7.0, and 9.0. For this, 50 μL of each MWCNT suspension was mixed with 5 mL of 1 mM KCl solution, also prepared at these pH values, to obtain a final concentration of 1.6 × 10⁻⁵ mg/mL of all MWCNT samples.

Nanoparticles were synthesized by single microemulsion method. Dichloromethane (DCM) was used to dissolve the BA, Res, PLGA (50 mg), and PLGA-PEG-Mal in 2 mL of in specific proportions. An oil-in-water emulsion was formed by dropping the polymer solution dropwise into 2.5% polyvinyl alcohol (PVA) solution (4 mL). Then, the solution was submitted to a probe sonicator on an ice bath of 25% amplitude for 4 min (Branson S-450; Danbury, CT, USA). Then the solution was gradually added to water, and then the residual DCM is removed by rotary evaporation for 15 min. The solution was stirred for 4 h to make the NPs spherical. After stirring, centrifuged at 15,000 rpm for 15 min and poured off the supernatant. Resuspended the precipitate with deionized water and repeated centrifugation 3 times to purify the NP. Finally, the precipitate was suspended with a small amount of ionized water and added to the cryopreservation solution, which was stored at − 80 °C. Blank@NP and Dye-loaded NP were prepared in the same way.