Multistirrer

PLGA NMeds were formulated via the nanoprecipitation technique. First, PLGA and the PLGA-Cy5 conjugate were solubilised in 4 mL of acetone and vortexed (total polymer weight = 50 mg) (Advanced Vortex Mixer ZX3, Velp^® Scientifica). This solution was added dropwise into a beaker containing 12.5 mL of Pluronic^® F68 under magnetic stirring (Multistirrer, Magnetic Stirrer Velp^® Scientifica, Usmate Velate, Italy) for 2 h at room temperature. The NMed suspension was purified by centrifugation at 9700 rpm for 10 min and resuspended in 4 mL of Pluronic^® F68 1.5% w/v. The NMed suspensions were stored at 4 °C for further analysis.
This general method was used to optimise the amount of PLGA-Cy5, ranging from 0.1–4% w/w of total PLGA. Further optimisations were then performed by maintaining the amount of PLGA-Cy5 constant at 0.2% w/w while varying the amount of Pluronic^® F68 concentration in the aqueous phase (12.5 mL) from 0–3% w/v. Finally, as previously indicated, the purified by centrifugation and the NMeds were resuspended in 4 mL of 1.5% w/v Pluronic^® F68.

NPs were produced following the nanoprecipitation protocol adapted from previously published literature [51 (link),52 (link)]. Briefly, 17 mg PLGA, 1 mg Cy5-labeled PLGA, and 2 mg DOTAP were dissolved in 2 mL of acetone and were added dropwise to 20 mL of poloxamer 188 0.5% w/v. The organic phase was evaporated for 2 h under magnetic agitation (Multi-Stirrer, Magnetic Stirrer Velp^® Scientifica, Usmate Velate, Italy). The NPs were then centrifuged at 9,700 RPM for 10 min to remove excess surfactant (ALC multispeed centrifuge PK 121, Camlab, Cambridge, UK) and the pellet was resuspended in MilliQ and stored at 4 °C until further use. Purified NPs were lyophilized (LyoLab 3000, Heto-Holten, ThermoFisher Scientific, Waltham, MA, USA) in pre-weighed Eppendorf tubes, and the weight yield% (WY%) was calculated as follows: