584 Vol. 43, No. 4 Biol. Pharm. Bull. 43, 584–595 (2020) © 2020 The Pharmaceutical Society of Japan Lipid Nanoparticles for Cell-Specific in Vivo Targeted Delivery of Nucleic Acids Ikramy A. Khalil, a,b Mahmoud A. Younis, a,b Seigo Kimura, a and Hideyoshi Harashima* ,a a Faculty of Pharmaceutical Sciences, Hokkaido University; Kita 12, Nish -6, Kita-ku, Sapporo 060–0812, Japan: and b Faculty of Pharmacy, Assiut University; Assiut 71526, Egypt. Received September 2, 2019 The last few years have witnessed a great advance in the development of nonviral systems for in vivo targeted delivery of nucleic acids. Lipid nanoparticles (LNPs) are the most promising carriers for producing clinically approved products in the future. Compared with other systems used for nonviral gene delivery, LNPs provide several advantages including higher stability, low toxicity, and greater efficiency. Additionally, systems based on LNPs can be modified with ligands and devices for controlled biodistribution and inter- nalization into specific cells. Efforts are ongoing to improve the efficiency of lipid-based gene vectors. These efforts depend on the appropriate design of nanocarriers as well as the development of new lipids with im- proved gene delivery ability. Several ionizable lipids have recently been developed and have shown dramati- cally improved efficiency. However, enhancing the ability of nanocarriers to target specific cells in the body remains the most difficult challenge. Systemically administered LNPs can access organs in which the capil- laries are characterized by the presence of fenestrations, such as the liver and spleen. The liver has received the most attention to date, although targeted delivery to the spleen has recently emerged as a promising tool for modulating the immune system. In this review, we discuss recent advances in the use of LNPs for cell- specific targeted delivery of nucleic acids. We focus mainly on targeting liver hepatocytes and spleen immune cells as excellent targets for gene therapy. We also discuss the potential of endothelial cells as an alternate approach for targeting organs with a continuous endothelium. Key words gene delivery; targeting; lipid nanoparticle; liver; spleen; endothelium 1. INTRODUCTION Gene therapy is defined as the use of different nucleic acids to express, edit, or silence specific genes in cells for obtaining specific therapeutic effects. The first nucleic acid sequences used in gene therapy were in the form of plasmid DNA (pDNA). More recently, RNA-based drugs have been in- troduced such as small interfering RNA (siRNA) and mRNA. Despite the great promise of gene therapy as a tool for treat- ing various currently incurable genetic as well as acquired diseases, it faces several difficulties such as the toxicity of viral vectors used in delivery of nucleic acids and the inef- ficiency of nonviral or synthetic vectors designed to replace the viral ones. 1–3) The inefficiency of nonviral systems could be explained by the lack of efficient delivery systems that suf- ficiently protect nucleic acids and deliver them in sufficient doses to their target sites, usually inside cells. 2,4) However, the last few years have witnessed a great advance in the de- velopment of nonviral systems for in vivo targeted delivery of genes to different organs, particularly the liver. This advance was translated into the approval of the first RNA interference (RNAi)-based drug, Patisiran (Onpattro), for the treatment of hereditary transthyretin-mediated amyloidosis (hATTR). 5–7) Patisiran is a lipid-based formulation encapsulating siRNA and efficiently targets liver hepatocytes after intravenous administration to cause efficient and durable silencing of the abnormal form of the transthyretin gene. This breakthrough in the field of nonviral gene delivery received substantial interest worldwide and clearly showed the importance of lipid-based systems for developing more approved drugs in the future. Several synthetic vectors are available for gene delivery. One strategy depends on using conjugates of different nucleic acids with different functional devices such as peptides, polymers, sugars, proteins, antibodies, or aptamers. 8,9) An- other strategy depends on encapsulating the nucleic acids in nanoparticles (NPs) of appropriate size. Conjugate systems are small in size and can easily be eliminated from the body through glomerular filtration in the kidney. 10) Furthermore, the nucleic acids in the conjugates are not protected and must be chemically modified to resist degradation with nucleases in the circulation. NP systems, on the other hand, are large enough to bypass kidney elimination and can provide more protection of nucleic acids in the circulation. The NPs used for gene delivery can be broadly classified into polymeric and lipid nanoparticles (LNPs). The latter provide several ad- vantages including higher stability, low toxicity, and greater efficiency. 11–14) In addition, systems based on LNPs can be easily modified with other ligands and devices for controlled biodistribution and internalization into specific cells. To increase the efficiency and decrease the side effects of lipid-based gene vectors, the systems must avoid rapid clear- ance and inactivation in the serum and must have the ability * To whom correspondence should be addressed. e-mail: harasima@pharm.hokudai.ac.jp Current Topics Recent Advances in Research on Particulate Formulations such as Lipoproteins, Liposomes, Extracellular Vesicles, and iPS-Derived Cells Review