Size-dependency of nanoparticle-mediated gene transfection: studies with fractionated nanoparticles

Abstract

Nanoparticles formulated from biodegradable polymers such as poly (lactic acid) and poly (d,l-lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their sustained release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated using an emulsion-solvent evaporation technique. However, this formulation procedure results in the formation of particles with heterogeneous size distribution. The objective of the present study was to determine the relative transfectivity of the smaller- and the larger-sized fractions of nanoparticles in cell culture. PLGA nanoparticles containing a plasmid DNA encoding luciferase protein as a marker were formulated by a multiple emulsion-solvent evaporation method (mean particle diameter=97±3 nm) and were fractionated using a membrane (pore size: 100 nm) filtration technique. The particles that passed through the membrane were designated as the smaller-sized nanoparticles (mean diameter=70±2 nm) and the fraction that was retained on the membrane as the larger-sized nanoparticles (mean diameter=202±9 nm). The smaller-sized nanoparticles showed a 27-fold higher transfection than the larger-sized nanoparticles in COS-7 cell line and a 4-fold higher transfection in HEK-293 cell line. The surface charge (zeta potential), cellular uptake, and the DNA release were almost similar for the two fractions of nanoparticles, suggesting that some other yet unknown factor(s) is responsible for the observed differences in the transfection levels. The results suggest that the particle size is an important factor, and that the smaller-sized fraction of the nanoparticle formulation predominantly contributes towards their transfection.

Introduction

Gene therapy, the introduction of an extraneous gene into a cell with the aim of replacing a lost cellular function or to introduce a new functionality, is fast becoming a reality (Rubanyi, 2001). However, achieving an efficient gene delivery into the target cell population or tissue without causing any vector-associated toxicity is critical to the success of gene therapy (Clark and Johnson, 2001). To achieve the above objective, various viral and non-viral vectors have been investigated (Kataoka and Harashima, 2001). Although viral vectors such as adenovirus, influenza virus and adeno-associated virus (Kochanek et al., 2001) are relatively more efficient in gene transfection than non-viral methods (Li and Huang, 2000), their toxicity and immunogenicity are the major concerns. Therefore, non-viral vectors such as liposomes, cationic block copolymers, polymer complexes, and micro- and nanoparticles have gained importance because they are relatively safe and are easy to formulate (Luo et al., 1999, Maheshwari et al., 2000). More recently, biodegradable nanoparticles formulated using different polymers such as chitosan (Mao et al., 2001, Roy et al., 1999), gelatin (Truong-Le et al., 1998), and other biodegradable polymers (Roy et al., 1999, Cohen et al., 2000) are being investigated as non-viral gene delivery systems because of the possibility of achieving safe and sustained gene transfection.

We have been investigating biodegradable nanoparticles formulated from poly (d,l-lactide-co-glycolide) (PLGA), an FDA approved biocompatible and biodegradable polymer, as a non-viral gene delivery system (Labhasetwar et al., 1999). Nanoparticles, because of their subcellular size, are effectively endocytosed by the cells which could result in higher cellular uptake of the entrapped DNA (Panyam et al., 2002b, Davda and Labhasetwar, 2002). Recently, we have demonstrated the rapid escape of PLGA nanoparticles from the endo-lysosomal compartment into cytoplasm, suggesting the suitability of nanoparticles as a gene delivery vector (Panyam et al., 2002b). Since the DNA is encapsulated inside the polymeric matrix, it would be protected from extracellular and intracellular nuclease degradation (Hedley et al., 1998). The DNA entrapped in nanoparticles is released slowly with the hydrolysis of the polymer matrix due to the cleavage of the ester bonds. It is hypothesized that the slow release of DNA from nanoparticles intracellularly would be effective in achieving sustained gene expression in the target tissue. Sustained and regulated gene expression is probably more important in treating certain localized disease conditions such as the cardiac and limb ischemia by inducing neovascularization in the damaged tissue using genes encoding pro-angiogenic growth factors (Richardson et al., 2001). Similarly, sustained gene expression has been shown to be effective in bone regeneration, which could be useful to repair fractured bones (Bonadio et al., 1999). Restenosis, a vasculoproliferative condition that occurs following coronary balloon angioplasty procedure, is another example of a pathological condition where sustained gene expression in the target artery could be more effective (Ohno et al., 1994, Klugherz et al., 2000).

Various formulation factors and characteristics of nanoparticles could influence the transfectivity of nanoparticles. One of the important parameters that could affect the transfectivity of nanoparticles is their size. The particle size has been an important consideration while formulating other particulate type systems such as DNA-polymer (Dauty et al., 2001) and lipid complexes (Lee et al., 2001) and liposomes (Sakurai et al., 2000). Our studies and that of others have shown that the particle size significantly affects their cellular and tissue uptake (Desai et al., 1997, Zauner et al., 2001), and in some cell lines, only the submicron size particles are taken up efficiently but not the larger size microparticles (e.g. Hepa 1-6, HepG2, and KLN 205) (Zauner et al., 2001). Nanoparticles prepared by emulsion-solvent evaporation technique using PLGA polymer usually results in the formation of nanoparticles with heterogeneous particle size distribution. We hypothesized that the transfectivity of the different sized nanoparticle fractions in the formulation could be different. Therefore, the objective of the present study was to determine the relative transfectivity of the smaller- and the larger-sized fractions of nanoparticles in cell culture.