Polymeric Core-Shell Nanoparticles Prepared by Spontaneous Emulsification Solvent Evaporation and Functionalized by the Layer-by-Layer Method

The aim of our study was to develop a novel method for the preparation of polymeric core-shell nanoparticles loaded with various actives for biomedical applications. Poly(caprolactone) (PCL), poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles were prepared using the spontaneous emulsification solvent evaporation (SESE) method. The model active substance, Coumarin-6, was encapsulated into formed polymeric nanoparticles, then they were modified/functionalized by multilayer shells’ formation. Three types of multilayered shells were formed: two types of polyelectrolyte shell composed of biocompatible and biodegradable polyelectrolytes poly-L-lysine hydrobromide (PLL), fluorescently-labeled poly-L-lysine (PLL-ROD), poly-L-glutamic acid sodium salt (PGA) and pegylated-PGA (PGA-g-PEG), and hybrid shell composed of PLL, PGA, and SPIONs (superparamagnetic iron oxide nanoparticles) were used. Multilayer shells were constructed by the saturation technique of the layer-by-layer (LbL) method. Properties of our polymeric core-shell nanoparticle were optimized for bioimaging, passive and magnetic targeting.


Optimization of the nanoemulsion's formation stabilized by AOT/PLL interfacial complex
For determination of the optimal oil phase/polycation ratio, the concentration of PLL was varied from 10 to 250 ppm, while a volume of the oil phase was fixed. The optimal amount of PLL was determined by measuring the zeta potential of formed nanodroplets and it corresponds to the point just before reaching the plateau of dependence of the zeta potential on the added amount of PLL. The optimal volume is marked by an asterisk ( Figure S2) indicating conditions when the unadsorbed PLL in the polymeric nanoparticles' suspension was minimized, as most of it was used to form the AOT/PLL interfacial complex. Figure S2. Changes of the zeta potential of emulsion cores with the AOT/PLL ratio. PCL/C-6 aq.
PLGA/C-6 aq. filtrate 4 the zeta potential on the added volume of PLL solution. The optimal volume is again marked by an asterisk ( Figure S4B) and it indicates the conditions when the zeta potential of formed polymeric core-shell nanoparticles reached a value, close to the zeta potential of the PLL in solution. The procedure described above was repeated until desired number of layers was formed. Optimized volumes of PGA and PLL used for the formation of consecutive layers of the shell were as follows: 1 ml PGA, 1.2 ml PLL, 1.5 ml PGA, 3.3 ml PLL, 4.0 ml PGA (polyelectrolytes' concentration 2 mg/ml).

PGA-g-PEG
For the preparation of pegylated polymeric core-shell nanoparticles, positively charged PLLterminated polymeric core-shell nanoparticles were coated with previously synthesized pegylated polyanion PGA-g-PEG [1]. The saturation concentration of PGA-g-PEG was determined using the same procedure as for a regular polyelectrolyte. The optimal volume of PGA-g-PEG used to form the stable pegylated external layer corresponds to the point just before reaching the plateau of dependence of the zeta potential on the added amount of PGA-g-PEG and is marked by an asterisk in Figure S5. The optimized volume of PGA-g-PEG was 17.5 ml (PGA-g-PEG concentration 200 mg/ml).  Figure S5. Changes of the zeta potential during formation of the PGA-g-PEG layer. The condition marked by an asterisk represents the first stable sample after overcharging.

Determination of saturation concentration for PGA and SPIONs
The PGA layer was built as described above, while the next layer in the shell structure was built from SPIONs (Superparamagnetic Iron Oxide Nanoparticles) using a procedure similar to that described for the polyelectrolyte. The fixed volume of PGA-terminated polymeric core-shell nanoparticles was added to SPIONs' suspension during continuous shaking. The volume of SPIONs' suspension used to form the saturated layer was chosen empirically and it corresponds to the point just before reaching the plateau of dependence of the zeta potential on the added volume of SPIONs. The optimal volume of SPIONs is again marked by an asterisk on Figure   S6 indicating conditions when the zeta potential of formed polymeric core-shell nanoparticles reached the value close to the zeta potential of SPIONs nanoparticles in solution. By this method, the amount of non-adsorbed SPIONs in the polymeric core-shell nanoparticles' suspension was minimized. Next hybrid layers were formed by repeating the procedure

Determination of saturation concentration for PGA and PLL-ROD
The PGA layer was built as described above. Then, the next layer in the shell structure was built from fluorescently-labeled polycation PLL-ROD. Fixed volume of PGA-terminated polymeric core-shell nanoparticles was added to PLL-ROD solution under mixing. The volume of PLL-ROD solution used to form the saturated layer was chosen empirically and it corresponds to the point just before reaching the plateau of dependence of the zeta potential on the added volume of PLL-ROD solution. The optimal volume is again marked by an asterisk ( Figure S7) indicating conditions when the zeta potential of formed polymeric core-shell nanoparticles reached the value close to the zeta potential of the PLL-ROD in solution. This procedure was repeated until the desired number of layers was formed. The optimized volumes of PGA and PLL-ROD used for the formation of consecutive layers of the shell were as follows: 1 ml PGA, 2 ml PLL-ROD, 1.5 ml PGA, 3 ml PLL-ROD, 4.5 ml PGA (all polyelectrolytes' concentration 2 mg/ml).