## 3 Results and Discussion  
### Preparation of DOX-NPs (7)  
Following a previously described protocol, a 500 nm monodisperse population of polystyrene amino-functionalized NPs (**1**) with 2% divinylbenzene (DVB) crosslinking were synthesized by dispersion polymerization (using 4-vinylbenzylamine hydrochloride- VBAH as the monomer to functionalize the nanoparticle with the amino groups) [25]. Following an Fmoc solid phase protocol, aminomethyl NPs (**1**) were double PEGylated (**4**) (100% yield). The density of PEGylation was calculated based on the amount of amino groups on the nanoparticle, this value being \(3.57\times 10^{-9}\) nmoles PEG/NP. The PEGylation increases the biocompatibility of the NPs, thereby facilitating their transport across cell membranes. It also reduces unfavorable interactions between NPs and the bioactive cargoes. Then, drug loading was carried out. The drug of choice was DOX due to its broad clinical use and it being one of the most effective chemotherapeutics for treating cancers, such as lung, breast, gastric, sarcoma, and pediatric cancers [29; 30; 31]. The designed strategy for the conjugation of DOX was via hydrazone bond. For this purpose, carboxylated NPs (**5**) were prepared using succinic anhydride; then, hydrazine functionalized NPs (**6**) were prepared, and the selective conjugation to the keto group in position C-13 of doxorubicin was carried out to yield DOX-NPs (**7**) (Scheme 1). Characterization of Drug-Loaded Nanoparticles (DOX-NPs) (**7**)  
The size distribution and zeta potential of the nanoparticles loaded with doxorubicin, DOX-NPs (**7**), together with amino functionalized nanoparticles, NAKED-NPs (**1**) were determined quantitatively by DLS (Figure 1a,c). The results obtained show a hydrodynamic diameter of \(464.2\pm 0.9\) nm with a PDI of 0.047, demonstrating that nanoparticle population was monodisperse (Figure 1a). The size was corroborated by TEM analysis (Figure 1d). The zeta potential value of DOX-NPs (**7**) was slightly negative (\(-13.7\) mV \(\pm\) 0.9) in water, as compared to NAKED-NPs (**1**), which was \(+81.2\) mV \(\pm\) 0.8, and Hydrazine-NPs (**6**)) which was \(+31.4\) mV \(\pm\) 0.3. (Figure 1c)). Keeping in mind that it has been described that to maintain the stability through electrostatic repulsion, zeta potentials of a particle should be above \(-20\) mV, it could be predicted that the stability of these particles could be compromised. However, we will like to highlight that these particles are stable. A stability assay has been runned (see Figure 1b) and DOX-NPs (**7**) were stable for six months at 4 \({}^{\circ}\)C and 25 \({}^{\circ}\)C. As the nature and number of molecules coated on the surfaces can affect the stability of polymeric particles, we could suggest that the balance between the loaded drug and the PEGylation on the surface has been a positive effect on the stability of these nanoparticles. Figure 1: Characterization of the DOX-NPs (**7**). (**a**) Hydrodynamic diameter of DOX-NPs (**7**) versus Naked-NPs (**1**); (**b**) Stability of DOX-NPs (**7**) for six months at 4 \({}^{\circ}\)C and 25 \({}^{\circ}\)C; (**c**) Values of the zeta potential of DOX-NPs (**7**) (red) versus Hydrazine-NPs (**6**) (green) and NAKED-NPs (**1**) (blue); (**d**) Transmission electron microscopy (TEM) analysis; (**e**) Dot plots representative of flow cytometry of NAKED-NPs (**1**) (blue) and DOX-NPs (**7**) (red); (**f**) Images of confocal laser microscopy of naked nanoparticles and DOX-NPs (**7**). Images with an increase of 63x show the signal of red channel. Scale bar, 10 \(\upmu\)msignal of red channel. Scale bar, 10 \(\upmu\)m. (**g**) Cumulative DOX Release. DOX-NPs (**7**) were incubated in phosphate solution at pH 6 and PBS at pH 7.4 for 168 h at 37 \({}^{\circ}\)C.The results are expressed with the values of the mean \(\pm\) SEM. The effect of storage conditions on the long-term stability of the NPs was investigated. The stability of DOX-NPs (7) was evaluated by DLS (Figure 1b).