## 2 Materials and Methods  
Eight surfactants--Tween 20 Sorbitanmonolaurate (MW 1228), Tween 40 Sorbitanmonopalmitate (MW 1277), Tween 80 Sorbitanmonooleate (MW 1310), Kolliphor P188 PPO-PEO-PPO triblock copolymer (MW, 7680-9510), Brij 010 Polyoxyethylene(10)oleyl ether (MW 709), Poly(vinyl alcohol)s (PVAs) such as Mowiol 4-88, MW 31,000 of 86.7%-88.7% hydrolyzed and Mowiol 8-88, MW 67,000 of 86.7%-88.7% hydrolyzed, purchased from Sigma-Aldrich (St. Louis, MO, USA), and Tritton X100 Poly(ethylene glycol)-p(1,1,3,3-tetramethyl-butyl)-phenyl ether (MW 647) purchased from Ferak Berlin GmbH (Berlin, Germany), were used as received. Organic solvents--1,3-Dimethyl-2-imidazolione (DMI), \(N\),\(N\)-Dimethylformamide (DMF), acetone and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) were purchased from Sigma-Aldrich, and Dimethylsulfoxide (DMSO) from [PERSON] (Karlsruhe, Germany). All the solvents were used as received. The dialysis bag (MWCO 25 kDa) was purchased from Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA. The AABB polymers, selected for the proposed study (Figure 1), were originally synthesized as reported previously [23; 30]--The PEUs **1F6** and **1L6** and the PEA **8L6** via the Interfacial Polycondensation (IP), and the PEA **4F4** via the Solution Active Polycondensation (SAP). The biodegradable cationic AABB polymer-arginine (**R**) based PEA **8R6** (Figure 2) was synthesized as reported previously using SAP [41].  
The number-average (\(M_{n}\)), and weight-average (\(M_{w}\)) molecular weights (MW), and molecular weight distribution (MWD) of the polymers were determined using the GPC. The MWs of the neutral polymers **1F6**, **1L6**, **4F4** and **8L6** were determined on a machine of Waters Associates, Inc., Milford, MA, USA, equipped with Styragel columns in DMF: HR4, HR3, HR0.5 all 7.8 mm \(\times\) 300 mm), a high-pressure liquid chromatography pump (Waters 1525 Binary HPLC) and a Waters refractive index detector 2414 and UV-detector (Waters 2487 dual absorbance detector, \(\lambda\) = 240 nm, Waters Associates, Inc., Milford, MA, USA). The polymers was dissolved in and eluted with DMF containing LiBr (0.1 M). The injected volume was 100 \(\mu\)L, the sample concentration was 5.0 mg/mL, and the flow rate was 1.0 mL/min. The columns were calibrated with PMMA standards. The MW of the cationic polymer **8R6** was determined on a Shimadzu GPC machine, model LC-8A equipped with an  
Figure 1: The chemical structures of the neutral AABB polymers used for fabricating the NPs.  
Figure 2: The chemical structure of the cationic AABB polymer **8R6** used for fabricating the positively charged NPs.  
Empower computer program (Waters), a PL HFIP gel column (Polymer Lab, Theale, Berkshire, UK) and a refractive index detector (Shimadzu RID-10A, Shimadzu Scientific Instruments, Columbia, MD, USA). The polymer was dissolved in and eluted with HFIP containing CF\({}_{3}\)COONa (0.05 M, to suppress polyelectrolyte effects). The injected volume was 100 \(\mu\)L, the sample concentration was 2.0 mg/mL, and the flow rate was 0.5 mL/min. The columns were calibrated with PMMA standards.  
Four different established cell lines (two murine, two human) were used: HeLa--Human epithelial cells from cervical cancer (ATCC CCL-2 TM, Manassas, VA, USA), A549--Human alveolar epithelial type II cells derived from lung carcinoma (ATCC CCL-185 TM, Manassas, VA, USA), Hepa1-6--Mouse hepatoma-derived cells (ATCC CRL-1830 TM, Manassas, VA, USA), and RAW264.7--Mouse leukemic monocyte macrophage cell line (ATCC TIB-71 TM, Manassas, VA, USA). All four cell lines (HeLa, A549, Hepa 1-6, and RAW264.7) are adherent and have be maintained in Dulbecco's Modified Eagle's Medium (DMEM) culture medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units of penicillin/mL, and 100 \(\mu\)g of streptomycin/mL at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) incubator. Cells were harvested with trypsin (0.25%)/Na\({}_{2}\)EDTA (Ethylenediaminetetraacetate disodium salt) solution.