PEGYLATED CHITOSAN- PLASMID DNA NANOPARTICLES: I) EFFECT OF PEGYLATION ON DRUG LOADING AND DRUG RELEASE

University: Biology

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Therefore, the objectives of the present work were; first to obtain and characterize nanoparticles made of chitosan chemically modified with PEG and, second, to evaluate the effect of pegylation on the in vitro behaviour of the resulting PEG modified chitosan:pDNA nanoparticles.

Materials and Methods

Materials

Chitech® M-010; low molecular weight chitosan (degree of deacetilation 89 %) was obtained from medicarb pSV--Galactosidase vector (model pDNA: 6820 bp) was purchased from Promega and PEG (Mw: 2000) was obtained from Merck. All chemicals were used as received and without further purification.

Preparation of Nanoparticles

Chitosan:DNA nanoparticles were prepared by complex coacervation method.

PEG-Chitosan:pDNA nanoparticles:

A chitosan solution were incubated for 24 hours with PEG aqueous solution (2% w/v). Then, the mixture was filtered from 5.000 MWCO (Vivaspin, Sartorius). Supernatant (500 L) was, then, gradually dropped into the pDNA solution (250 L) during vortex at the highest speed for 30 sec. (Figure 1).

PEG coated Chitosan:pDNA nanoparticles:

Solutions of chitosan and pDNA were separately prepared. A chitosan solution and a DNA solution of 100 g/mL in 50 mM sodium sulfate solution were preheated to 55C. Chitosan solution (500 L) was, then, gradually dropped into the pDNA solution (250 L) during vortex at the highest speed for 30 sec. After nanoparticles optained, PEG aqueous solution (0.5% w/v) were incubated for 24 h with nanoparticles (Figure 1).


Figure 1: Schematic presentation of the nanoparticle preparations

Characterization of Nanoparticles

Measurements of particle size and the zeta potential measurements were performed using an aqueous dip cell in the automatic mode by Zetasizer Nano ZS (Malvern Instruments). The pDNA loading capacity of nanoparticles and the pDNA encapsulation efficiency of the processwere determined by ultracentrifugation of the samples. The amount of free pDNA was determined in a clear supernatant by spectrophotometry at 260 nm. The amount of pDNA encapsulated in the nanoparticles was calculated by measuring the difference between the total amount of pDNA added in the nanoparticle preparation and the amount of non-entrapped pDNA remaining in the aqueous suspension. The pDNA loading capacity was calculated from equations as described by Bozkir and Saka (2004). The morphological examination of nanoparticles was observed by transmission electron microscope (LEO 906 E).

Agarose Gel Electrophoresis

The DNA binding ağabeylity of chitosan or its PEGylated derivatives were evaluated by agarose gel electrophoresis. The complexes containing 0.5 µg of pDNA were loaded into individual wells of 1.0% agarose gel in 1x Tris-boric acid- EDTA buffer, electrophoresed at 50 V for 4 hours, and stained with 0.5 µg/mL ethidium bromide. The resulting DNA migration pattern was revealed under UV irradiation.
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