Hyaluronic acid controls the uptake pathway and intracellular trafficking of an octaarginine-modified gene vector in CD44 positive- and CD44 negative-cells
Introduction
The cellular uptake pathway of a gene vector is a major factor in transgene expression [1], [2], [3], [4], [5]. We previously reported that carriers with a high-density of octaarginine (R8), an artificially designed cell penetrating peptide [6], [7], were efficiently internalized by cells primarily via macropinocytosis rather than clathrin-mediated endocytosis, as is the case for cationic liposomes (LPs) [8]. We also developed an R8-modified gene vector, a multifunctional envelope-type nano device (MEND), consisting of a condensed plasmid DNA (pDNA) core and lipid envelopes [9], [10]. The integration of R8 into the MEND (R8-MEND) dramatically enhanced the transfection activity of the MEND, approaching values as high as that for adenovirus [9], [11], while the R8 needs to overcome its poor cell selectivity, since it facilitates the cellular uptake nonspecifically regardless of the cell type.
To date, our group has reported that a dual-ligand liposomal system comprised of a specific ligand and a cell-penetrating peptide (CPP) enhanced both selectivity and cellular uptake efficiency [12], [13]. Takara et al. prepared dual-ligand PEGylated liposomes by modifying the end of the polyethylene-glycol (PEG) with an NGR (Asn-Gly-Arg) tumor neovasculature-homing motif peptide, which recognizes CD13, a marker for tumor endothelial cells, followed by coating the surface of the liposomes with CPP [12]. The dual-ligand system stimulated the uptake of the carriers by CD13 positive cells, synergistically [12]. Kibria et al. also reported that dual-ligand PEGylated R8-modifeid liposomes with the cyclic RGD (Arg-Gly-Asp) peptide, a specific ligand for Integrin αvβ3, showed an enhanced cellular uptake as well as a higher transfection efficiency in Integrin αvβ3 expressing cells [13]. These reports indicate that a dual-ligand liposomal system can be a useful strategy for achieving cell selective targeting with a high transgene expression.
More recently, we developed a dual-ligand positively charged R8-MEND that was modified with negatively charged hyaluronic acid (HA) via electrostatic interactions but not with PEG [14]. HA is a natural ligand for cancer cells and liver endothelial cells overexpressing CD44, thus it would be expected that HA would be a specific ligand for/targeted to cancer and liver endothelial cells. In that study, we investigated the transgene expression of an R8-MEND modified with HA in liver endothelial cells (liver ECs) (CD44 positive cells), and determined the optimal composition of MEND needed for efficient transgene expression in liver ECs, which possesses an HA-coated lipid envelope modified with the R8 [6], [7] and GALA, a pH-sensitive fusogenic peptide for efficient endosomal escape [15], [16] (HA-R8/GALA-MEND).
The focus of this study was on the mechanism responsible for the cellular uptake pathway and transgene expression of the HA-R8/GALA-MEND. A series of R8/GALA-MENDs coated with various concentrations of HA (600 kDa or 80 kDa) were prepared and the physicochemical properties and the transfection activity of these preparations in HCT116 cells overexpressing CD44 were evaluated. We then investigated the cellular uptake pathway of the HA-R8/GALA-MEND and the R8-MEND using HCT116 cells, and determined the optimal composition of HA-R8/GALA-MEND required for efficient cellular uptake via a CD44-mediated pathway. Moreover, we compared the cellular uptake pathway and transfection activity of HA-R8/GALA-MEND between HCT116 cells and NIH3T3 cells (CD44-negative cells). Based on these results, we analyzed the relationship between the cellular uptake pathway and transgene expression in HCT116 cells and NIH3T3 cells. A model for transgene expression via a CD44-mediated pathway is proposed in an attempt to understand the intracellular fate of HA-coated gene vectors.
Section snippets
Materials
The pcDNA3.1 (+)-luc plasmid was constructed by inserting the firefly luciferase gene (HindIII–XbaI fragment) of the pGL3-Control plasmid (Promega, Madison, WI, USA) into the pcDNA 3.1 (+) plasmid containing cytomegalovirus promoter (Invitrogen, Carlsbad, CA, USA) pretreated with the same restriction enzymes. The pDNA was purified using a Qiagen EndoFree Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). Amiloride and cholesteryl hemisuccinate (CHEMS) was purchased from Sigma (St. Louis, MO,
Preparation of HA-R8/GALA-MEND
A series of R8/GALA-MEND coated with various concentrations of HA (600 kDa or 80 kDa) was prepared as shown in Fig. 1. To construct the R8/GALA-MEND, condensed pDNA particles were packaged with a lipid envelope modified with the R8 peptide, a cellular uptake device [7], [8], [11], [17], and GALA, a pH-sensitive fusogenic peptide [15], [16]. In the preparation, HA coats the carrier surface via electrostatic interactions between the positively charged R8/GALA-MEND (∼+50 mV) and the negatively
Discussion
In this study, we prepared an HA-R8/GALA-MEND, which possesses a lipid envelope modified with R8 and GALA in which the surface is coated with HA. In the preparation, HA coats the carrier surface, when the R8/GALA-MEND and HA are simply mixed with one another. The coating is held in place via electrostatic interactions and does not involve PEGylation, since PEGylation typically results in a decrease in the transfection activity of the carrier, as previously reported [14]. An HA-R8/GALA-MEND
Conclusion
In this study, we analyzed the cellular uptake pathway involved in the gene expression of an HA coated gene vector (HA-R8/GALA-MEND) using CD44 positive and negative cells. The findings indicate that the HA (600 kDa)-R8/GALA-MEND was efficiently taken up by cells and that transfection occurred via macropinocytosis and a CD44-medeiated pathway in HCT116 cells overexpressing CD44. While, in the case of NIH3T3 (CD44 negative) cells, the HA (600 kDa)-R8/GALA-MEND was internalized mainly via
Acknowledgments
This study was funded by a Grant-in-Aid for Young Scientists (A) [Grant No. 23680053 (Y.Y.)], a Grant-in-Aid for Scientific Research (B) [Grant No. 26282131 (Y.Y.)] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), and in part by the Program for Promotion of Fundamental Studies in Health Sciences [Project ID. 10-62 (Y.Y.)] of the National Institute of Biomedical Innovation, Japan (NIBIO). We also thank Dr. Milton Feather for his helpful
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These authors contributed equally as first author.