Investigation of endocytosis and cytotoxicity of poly-d, l-lactide-poly(ethylene glycol) micro/nano-particles in osteoblast cells

Abstract

Biodegradable polymer particles present a promising approach for intracellular delivery of drugs, proteins, and nucleic acids. Poly-d,l-lactide-poly(ethylene glycol) (PELA) copolymers with different weight ratios of polyethylene glycol (PEG) were used as drug carriers in the present study. PELA particles entrapped with fluorescein isothiocyanate (FITC) as a fluorescent marker were formulated using a double emulsion-solvent evaporation technique. The size and morphology of the particles were observed with scanning electron microscope (SEM), atomic force microscope (AFM), and laser diffraction particle size analyzer (LDPSA). The purpose in the present work was to investigate the cytotoxicity and the process of endocytosis of PELA particles with different contents of PEG and variable particle size using rat osteoblasts (OBs). The cytotoxicity of the particles was investigated using 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Results indicate that as the content of PEG in the polymer increased, so did cell survival. Endocytosis was observed through a light microscope and a fluorescence microscope; intracellular uptake and retention were determined quantitatively using fluorescence spectrophotometer (FSP). The results showed that as PEG content in PELA copolymer increased, there was a reduction in endocytosis of nanoparticles in osteoblasts.

Keywords: cytotoxicity; drug delivery; endocytosis; intracellular trafficking; nanoparticles.

Figure 1

A) Summary of characterization of particles. B) Size distribution detected by laser diffraction particle size analyzer.

Figure 2

Morphology of particles detected by SEM. A, B, C, D, E, and F denote PLA-n, PELA10-n, PELA20-n, PELA30-n, PELA10-1, and PELA10-10, respectively.

Figure 3

Morphology of nanospheres detected by AFM. A, B, C, and D denote PLA-n, PELA10-n, PELA20-n, PELA30-n, respectively. Samples were prepared for AFM investigation.

Figure 4

A) Optical microscopic observation (×200) of osteoblasts incubated with PLA-n (A), PELA10-n (B), PELA20-n (C), PELA30-n (D), PELA10-1 (E), and PELA10-10 (F) particles from 1 to 3 days. B) Viability of osteoblasts from 1 to 3 days by MTT assay, P < 0.05.

Figure 5

Cell apoptosis profiles of osteoblasts as a function of incubation time (0.3 mg/mL, 37 °C) detected by flow cytometer A) and cell apoptosis rate of osteoblasts by flow cytometer B).

Figure 6

A) Fluorescence intensity of nanospheres in osteoblasts detected by fluorescence spectrophotometer. B) Fluorescent microscopy utilized to assess cellular uptake of nanospheres as a function of incubation time (0.3 mg/mL, 37 °C). Osteoblasts were incubated with the nanospheres under the condition as indicated above each image, and samples were prepared for fluorescence microscope (×200) investigation. Bars = 20 μm.

Figure 7

TEM photo showing intracellular fate of nanospheres. Some nanospheres were localized in endosomes and lysosomes. Abbreviations: Ly, lysosome; LE, late endosomes; EE, early endosomes; V, vacuole; CM, cell membrane; NP, nanoparticles.