양이온성 폴리스티렌 나노입자: 유전자 전달을 위한 모델 전달체의

제조 및 특성분석 (Ⅰ)

방시원, 박현영a, 장양수a 김중현*, 김우식

연세대학교 화학공학과, 연세대학교 의과대학 심혈관연구소a

Cationic Polystyrene Nanoparticles: Preparation and Characterization of a Model Carrier System for Gene Delivery (Ⅰ)

Shi-Won, Pang, Hyun-Young, Parka, Yangsoo, Janga, Jung-Hyun, Kim* and Woo-Sik Kim

Dept. of Chemical Engineering, Yonsei University

Yonsei Cardiovascular Research Institute, Collage of Medicine, Yonsei Universitya

Introduction

In recent years there has been a growing interest in the use of polymeric systems as carriers for gene substances. These systems can take the form of either soluble polymers or biodegradable microspheres.

In general terms, polymeric microspheres for gene delivery can be considered under the general classification as colloidal carriers, which includes, not only microspheres and nanoparticles, but also liposomes, emulsions and materials of biological origin such as low density lipoproteins. Cellular uptake of ODN, as well as their activity in cell cultures, can be improve by cationic liposomes [1-3], first used to improve cell transfection with plasmid DNA [4] and RNA [5]. Positively charged particles are prone to nonspecific interactions with plasma proteins and negatively charged cell surfaces. This may lead to destabilization, dissociation, and rapid clearance of the liposome-ODN complexes by the mononuclear phagocyte system before reaching the diseased tissue. It is generally accepted that adenoviral vectors are quite effective in vivo. However, safety issues and immunological responses are major concerns for repeated administration of viral vectors. Non-viral approaches in which plasmid is complexed with cationic lipids have been used to deliver genes to the lungs.

Experimental

Preparation and Characterization of the Nanoparticles

Polystyrene latexes and cationic polystyrene latexes were synthesized via batch polymerization under emulsifier-free conditions in aqueous medium using a 1.5-liter internally stirred double-jacket reactor at 70oC with a condenser, nitrogen bleed, and direct stirrer. Cationic amino-modified PS/DSDM was synthesized for the purpose of binding with genes. The shape and size of the nanoparticles were determined by scanning electron microscopy and the average particle diameters were determined by the capillary hydrodynamic fractionation from Matec Applied Science, USA. The number density of surface functional groups was determined by conductometric titration [6-8], where Metrohm auto titrator (Swiss, consists of 702 SM Tirtino, 703 Ti Stand, and 660 Conductometer) was used.

Toxicity of the Nanoparticle Suspensions

Cytotoxicity of the purified nanoparticles was determined that 293 cell, hela and MCF-7 were incubated with the various concentration of nanoparticle suspension in cell culture medium in 3 hr, washed the cell culture with PBS solution and checked the amount of the viability of cells after 48 hours.

Adsorption Isotherms

To investigate the adsorptive properties of the nanoparticles, it incubated polymer suspensions with the various concentration of DNA at 25oC in 1.0 ml PBS buffer (pH=7.4) over a period of 1hr. To determine the amount of adsorbed DNA on the latex, the particles were separated by centrifugation (15,000g, 30min and 5oC) and the DNA concentration in the supernatant was determined by fluorometrically by incubating 200□ of supernatant with 4□ of ethidium bromide solution (50□/ml) using a microplate spectrofluorometer at □ex = 490nm and □em = 590nm (LB 50B, Perkin Elmer, USA) .

Result and Discussion

Characterization and Cytotoxicity of Nanoparticles

Figure 1 shows the shape and size of nanoparticles. Figure 2 shows representative date from three different experiments per cell line. Thus, it is necessary to minimize the size of the particles for parental administration. Results show that PS and PS/DSDM nanoparticles do not influence the cell viability significantly. The cell viability of nanoparticles ranged between 75∼110% of control in all experiments. At a maximum nanoparticle concentration of 50□/ml, the mean cell viability of three different cell lines was 91±17% of control.

Adsorption Isotherms

The interaction of DNA with the nanoparticles was studied by the adsorption isotherms. To evaluate the influence of latex type on the adsorption, latexes bearing different  amounts of charged end groups were applied. The results showed in figure 3. The materials slightly differ in their loading capacity in relation to the amount of polymer. However, considering the amount of adsorbed DNA relative to the surface area, the values show variations that can be mainly attributed to differences in the surface charge densities of the latexes. Figure 3-(a) showed that the adsorption amount of DNA on the not-modified polystyrene particles was about 17□ on the latexes. Figure 3-(b) showed that the adsorption amount of DNA on the amino-modified polystyrene particles was about 22□. It was explained that the driving forces of the DNA adsorption existed at least two kinds of forces. The one was the hydrophobic interaction and the other was electrostatic one. The surface charge of DNA was negative, so particles with positive charge was likely to interact with the opposite charge ones. Taking into account the results from the adsorption studies, the adsorptive binding of DNAs appears to be essentially irreversible and localized due to multiple electrostatic interactions with the interface.

Reference

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