Expression of beta-galactosidase

The in vitro transfection ability of cationic nanoparticle of formula A, B, C and D with DNA complexes were evaluated in COS-7 and HeLa cell line using pCMV-βgal as a report gene. At 4 and 24 h after transfection, expression of beta-galactosidase was quantified enzyme assay, indication transfection. The cationic nanoparticles formulations have a higher transfection effect than PEI 25k alone for N/P ratio of 77/1 to 308/1 at 4h after transfection (Fig. 6A) and N/P ratio of 19/1 to 77/1 at

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24h after transfection in COS-7 and HeLa cells (Fig. 6B and 6C).

4. Conclusion

In this study, it is advantageous in making complexes combining PEI with PLGA by an emulsion- diffusion-evaporation technique using PVA as a stabilizer to form cationic nanoparticles showed that can bind DNA by electrostatic interaction. Effect of PLGA variables on nanoparticles formulations exhibited reduce to toxicity and enhance transfection efficiency in cells than PEI alone.

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Figure 1. Effect of PVA concentration on PLGA particle size (the PLGA concentration was fixed at 1mg/ml, n=3)

Particles SA Particles SB Particles SC

Particles SD

Table 1.Summary of formulation composition of nanoparticles and the corresponding SEM micrographs

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Table 2. Particle sizes and zeta potential of PLGA/PEI cationic particles (n = 3; PDI : Polydispersity index)

Figure 2. Effect of formula variables of carriers/DNA binding interaction was determined by gel electrophoresis. Lane 1, 400ng DNA (pCMV-βgal); Lanes 2-6, Cationic particles/pCMV-βgal (N/P ratio): 38/1, 19/1, 4/1, 1/1, 0.2/1

A 264.0±5.1 0.10±0.03 A 18.3±1.2

B 250.1±6.5 0.10±0.06 B 15.1±2.6

C 223.7±1.6 0.05±0.05 C 8.2±2.5

D 210.7±1.4 0.10±0.05 D 7.5±2.9

Formula A

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SA (a)

(b)

70

SB

SC

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SD

Figure 3. AFM images of nanoparticles in solution state (a) Formula B (b) Formula B/pCMV-βgal complexes (N/P = 38/1). And SEM micrographs of Formula A (SA), Formula B (SB), Formula C (SC) and Formula D (SD).

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Figure 4. Effect of Formula with variable N/P ratios on cytotoxicity of cationic particles/pCMV-β gal complexes analyzed by MTT assay, (A) 4h (B) 24h after transfection in COS-7 cells (n=3).

(A)