Galactosylated ternary DNA/polyphosphoramidate nanoparticles mediate high gene transfection efficiency in hepatocytes

Abstract

Galactosylated polyphosphoramidates (Gal-PPAs) with different ligand substitution degrees (6.5%, 12.5% and 21.8%, respectively) were synthesized and evaluated as hepatocyte-targeted gene carriers. The in vitro cytotoxicity of Gal-PPA decreased significantly with an increase in galactose substitution degree. The affinity of Gal-PPA/DNA nanoparticles to galactose-recognizing lectin increased with galactose substitution degree. However, decreased transfection efficiency was observed for these galactosylated PPAs in HepG2 cells. Based on the results of gel retardation and polyanion competition assays, we hypothesized that the reduced transfection efficiency of Gal-PPA/DNA nanoparticles was due to their decreased DNA-binding capacity and decreased particle stability. We therefore prepared nanoparticles by precondensing DNA with PPA at a charge ratio of 0.5, yielding nanoparticles with negative surface charge, followed by coating with Gal-PPA, resulting in a Gal-PPA/ DNA/PPA ternary complex. Such a ternary nanoparticle formulation led to significant size reduction in comparison with binary nanoparticles, particularly at low N/P ratios (2 to 5). In HepG2 cells and primary rat hepatocytes, and at low N/P ratios (2 to 5), transfection efficiency mediated by ternary nanoparticles prepared with 6.5% Gal-PPA was 6–7200 times higher than PPA-DPA/DNA nanoparticles. Transgene expression increased slightly at higher N/P ratios in HepG2 cells and reached a plateau at N/P ratios between 5 and 10 for primary rat hepatocytes. Such an enhancement effect was not observed in HeLa cells that lack of asialoglycoprotein receptor (ASGPR). Nevertheless, transfection efficiency of ternary particles decreased dramatically, presumably due to the decreased DNA binding capacity and particle stability, as PPA galactosylation degree increased. This highlights the importance of optimizing ligand conjugation degree for PPA gene carrier.

Introduction

Gene therapy promises new approaches for treating many inherited and acquired diseases. However, its full potential can not be realized until successful delivery systems become available [1]. An ideal gene delivery vector should be safe, stable, and cost-effective to produce in clinically relevant quantities, and capable of efficient and tissue-specific gene delivery. Viral vectors [2], [3] such as retroviruses and adenoviruses have been extensively investigated and demonstrate transfection efficiency relevant to clinical applications. However, their applications in clinical settings are limited because of safety concerns [4] and limitation on packaging sizes of genes. Cationic polymeric gene carriers, as non-viral carriers, offer promising alternatives. Cationic polymeric carriers mediate gene transfer by condensing DNA into nanoparticles, protecting DNA from enzymatic degradation, and facilitating cellular uptake and/or endolysosomal escape and/or nuclear translocation. A number of polycations have been reported to effect gene transfection, including poly-l-lysine (PLL) [5], [6], chitosan [7], [8], polyamidoamine dendrimers [9], polyethylenimine [10], [11], [12], poly[α-(4-aminobutyl)-l-glycolic acid] [13], [14], poly-l-glutamic acid derivatives [15], poly (β-amino esters) [16], [17]. Despite the extensive effort in synthesizing new polymeric carriers, an efficient polycation with low toxicity and good stability has not yet emerged.

Incorporating cell- or tissue-specific ligands to a polycationic carrier has been widely investigated as an effective approach to improve gene transfer efficiency in target tissue or cells. A number of ligands have been examined including galactose, transferrin, folate, RGD peptides and antibodies [18], [19], [20], [21], [22]. Among them, galactose is the most extensively studied to target genes to liver parenchymal cells since galactose moiety can be specifically recognized by asialoglycoprotein receptors (ASGPR) on hepatocytes. Gene transfer efficiency can be improved as a result of the enhanced cell uptake via ASGPR-mediated endocytosis. For instance, galactosylated polylysine (Gal-PLL) showed a more than 10-fold increase in transfection efficiency in HepG2 cells compared with unmodified PLL [23]. Gal-PLL/DNA complexes were preferentially taken up by liver parenchymal cells after intravenous injection [24]. In a study by Sagara et al. [25], galactosylated poly(ethylene glycol)-PEI showed 2-fold higher transfection efficiency than unmodified PEI.

Recently, we developed a series of phosphorus containing polymeric gene carriers with phosphoester linkage in the backbone [26], [27]. The unique characteristic of the pentavalency of the phosphorus atom makes it possible to conjugate functional groups in its side chains, including charged groups through a phosphate or phosphoramide bond, categorized into polyphosphoester and polyphosphoramidate, respectively. Both polyphosphoesters and polyphosphoramidates have demonstrated marked capabilities for DNA compaction and protection. Previous results have shown that polyphosphoester [28], [29] and polyphosphoramidate [30] with primary amino group side chains exhibit higher transfection efficiencies than those that carry secondary, tertiary or quaternary amino group side chains [30]. Polyphosphoramidate gene carriers bearing primary amino side chains with branching spacers are 7- to 10-fold more efficient than that with linear spacers (Fig. 1). Among all polyphosphoramidates with primary amino groups, those with N,N-bis (aminopropyl) amino side chains, for example, PPA-DPA (Fig. 1), showed similar transfection efficiency as PEI at a charge ratio of 10, but much lowered cytotoxicity in cell line. The LD50 of PPA-DPA is four times higher than PEI [31]. These results have motivated us to explore further improvement on transfection and biocompatibility of PPA-DPA (hereafter called PPA for short) based on the ligand conjugation approach.

In this study, we have synthesized three galactose-bearing carriers, Gal-PPAs, with different degrees of substitutions (6.5%, 12.5% and 21.8%) as hepatocyte-targeted gene carriers, aiming to improve gene transfer efficiency to the liver. We also explored two different schemes in preparing galactosylated PPA/DNA nanoparticles, taking advantage of the high DNA compaction capacity of PPA-DPA carrier. These two types of nanoparticles, namely, Gal-PPA/DNA binary nanoparticles and Gal-PPA/DNA/PPA ternary nanoparticles, were characterized for their physicochemical properties, cytotoxicity and transfection efficiency in cell lines and primary rat hepatocytes. The effects of formulation scheme, N/P ratio and the galactosylation degree on transfection efficiency of the particles were investigated.