A small variation in average particle size of PLGA nanoparticles prepared by nanoprecipitation leads to considerable change in nanoparticles’ characteristics and efficacy of intracellular delivery

Figures & data

Table 1. Independent variables and response for three-factor, two-level full factorial statistical experimental design.

Factors	Used levels
Independent variables	Low	High
Polymer amount (X1) (mg)	10	30
Emulsifier concentration (X2) (%)	0.5	1
Organic phase volume (X3) (ml)	1	3
	Response
Average particle size (nm)	Y

Table 2. Characteristics of PLGA₂₃₀ and PLGA₁₆₀ nanoparticles.

	Averageparticle size (nm)	Average particle size (nm) in cell culture medium after 24 h	PdI	Zeta potential (mV)	Encapsulationefficacy (%)	Dye amount innanoparticles (μg/mg)
PLGA160 nanoparticles	157.9 ± 6.16	152.3 ± 4.54	0.10 ± 0.04	−11.2 ± 4.7	25.7 ± 1.7	0.61 ± 0.03
PLGA230 nanoparticles	230.8 ± 4.32	210.2 ± 8.3	0.11 ± 0.03	−6.32 ± 0.9	57.1 ± 2.9	0.62 ± 0.04

Figure 1. Size and dye release properties of PLGA₂₃₀ and PLGA₁₆₀ nanoparticles. (A) Average particle size of Dil-labelled PLGA nanoparticles as a function of the polymer concentration. (B) Release of Dil from PLGA₂₃₀ and PLGA₁₆₀ nanoparticles into 0.5% (w/v) SDS containing PBS. AFM (C) and TEM (D) images of PLGA₂₃₀ nanoparticles and PLGA₁₆₀ nanoparticles.

Figure 2. Effect of different-sized PLGA nanoparticle counts (nanoparticle number/ml) on cell viability and cellular uptake. (A) Cytotoxicity of PLGA₂₃₀ and PLGA₁₆₀ nanoparticles incubated at different nanoparticles counts with HEK293 cells. Cytotoxicity was determined using DRAQ7 viability dye. Percentage of viable cells is shown. (B) Typical DRAQ7 viability assay flow cytometry contour plots for cells treated with 5 × 10¹⁰/ml PLGA₂₃₀ and PLGA₁₆₀ nanoparticles. Purple (or gray) contours represent dead cells that are labelled with DRAQ7 (FSC, forward scatter). (C) Percentage of Dil⁺cells that engulfed nanoparticles at the end of 24 h incubation time. (D) Change in the fluorescence intensity at various time points showing uptake kinetics of Dil-labeled PLGA₂₃₀ and PLGA₁₆₀ nanoparticles applied at counts number of 1.25 × 10¹⁰/ml. (E) Fluorescence microscopy images of PLGA₂₃₀ and PLGA₁₆₀ nanoparticles incubated with cells at same numbers of nanoparticles/ml (1.25 × 10¹⁰/ml). The cell nuclei were stained with DAPI. The Dil-loaded nanoparticles can be observed as inclusion bodies. (F) The fluorescence microscope images of the cells treated with same weight of PLGA₂₃₀ and PLGA₁₆₀ nanoparticles (G) The uptake efficiency of nanoparticles when the fluorescence intensity was normalized to the amount of dye encapsulated (i.e. weight of the nanoparticles) into the nanoparticles (1.25 × 10¹⁰/ml, 24 h incubation). (*p < 0.05).

Figure 3. Evaluation of endocytic pathways employed in cell uptake of PLGA₂₃₀ (A and B) and PLGA₁₆₀ (C and D) nanoparticles. The cells were pre-treated with different endocytosis inhibitors (as shown in B and D) and the same numbers of Dil-labeled nanoparticles were applied onto the cells. The percentage change in median fluorescence intensity values were calculated in comparison to the control cells that were not treated with inhibitors. Schematic representation of possible intracellular drug delivery mechanisms of PLGA₂₃₀ (B) and PLGA₁₆₀ (D) nanoparticles are summarized.