Cardiomyocyte uptake mechanism of a hydroxyapatite nanoparticle mediated gene delivery system.

Hiroaki Komuro,Akiko Nagai,Tetsuo Sasano,Masahiro Yamazoe,Kosuke Nozaki

doi:10.3762/bjnano.11.150

Hiroaki Komuro, Akiko Nagai + Show 3 more

Open Access

https://doi.org/10.3762/bjnano.11.150

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Abstract

Gene therapy has been explored as a future alternative for treating heart disease. Among several gene delivery systems aimed at penetrating specific target cells, we focused on safe and non-viral gene delivery materials with a high transfection efficiency. Although various techniques have been developed, the mechanisms underlying the cellular uptake of gene delivery materials have not yet been sufficiently studied in cardiomyocytes. The aim of this study was to determine how hydroxyapatite (HAp) nanoparticles contribute to the delivery of plasmid DNA (pDNA) into cardiomyocytes. We fabricated HAp nanoparticles using the water-in-oil (W/O) emulsion method and used these nanoparticles as the delivery vector for transfecting cardiomyocyte-derived HL-1 cells. HAp exhibited particles on the nanoscale and with a low cytotoxicity in HL-1 cells. The transfection assay performed with several endocytosis inhibitors suggested that the HAp/pDNA complexes were internalized by HL-1 cells through macropinocytosis. Furthermore, this HL-1 cell uptake was generated in response to HAp stimulation. Thus, HAp is a positive regulator of macropinocytosis in HL-1 cells and a good system for gene delivery in cardiomyocytes.

Highlights

Heart disease is one of the major causes of death and accounts for approximately one in every three deaths worldwide every year [1]
We previously demonstrated that the cellular uptake of HAp nanoparticles into endothelial cells (ECs) was mainly through caveolae-mediated endocytosis [22]
We show that HAp nanoparticles are vectors that effectively transfect cardiomyocytes in comparison to ECs and the uptake of HAp in cardiomyocytes is through macropinocytosis

Summary

Introduction

Heart disease is one of the major causes of death and accounts for approximately one in every three deaths worldwide every year [1]. Since gene therapy for heart disease will potentially become the standard treatment in the future, many studies using viral and non-viral vectors have been conducted in cardiomyocytes. Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) is one of the most stable forms of CaP, and diverse methods for preparing HAp nanoparticles have been reported Among these methods, the microemulsion method has the advantage of controlling the spherical-like morphology and the size of nanoparticles by using a surfactant solution [11,12]. We show that HAp nanoparticles are vectors that effectively transfect cardiomyocytes in comparison to ECs and the uptake of HAp in cardiomyocytes is through macropinocytosis This pathway can be activated by the stimulation of HAp. The HL-1 cells were used as the in vitro model of cardiomyocytes in this study [23]. Since HL-1 cells are derived from mouse atrial myocytes they have an adult cardiomyocyte phenotype with the expression of cardiac-specific functional receptors

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