**1. Introduction**

Nucleic acid vaccines are being evaluated for use in a number of clinical applications, including cancer, allergy, and infectious diseases [1–5]. It is particularly noteworthy that mRNA was first commercialized as a vaccine against the coronavirus disease 2019 (COVID-19) for the treatment of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [6–8], and its application to a wide variety of diseases is now being investigated. The mRNA molecule, unlike DNA, has no risk of unintentional insertion into genomic DNA [9]. In addition, mRNA does not require nuclear delivery and can efficiently express the encoded gene(s) if it is properly introduced into the cytoplasm. Recent developments in mRNA engineering, such as capping technology, sequence design technology, and chemically modified bases, as well as relatively simple synthesis by in vitro transcription, have greatly contributed to the development of this technology [10]. In addition, mRNA can express any peptide and protein structures depending on the sequence design, making it possible to present a variety of antigen peptides to major histocompatibility complex (MHC) classes I and II [11]. In addition, since mRNAs exhibit immunostimulatory properties through recognition by pattern recognition receptors (PPRs), including Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) families [12], which recognize pathogen-associated molecular patterns (PAMPs), they can activate innate immunity efficiently without the need for additional adjuvants [2,13].

**Citation:** Sasaki, K.; Sato, Y.; Okuda, K.; Iwakawa, K.; Harashima, H. mRNA-Loaded Lipid Nanoparticles Targeting Dendritic Cells for Cancer Immunotherapy. *Pharmaceutics* **2022**, *14*, 1572. https://doi.org/10.3390/ pharmaceutics14081572

Academic Editor: Jesus Perez-Gil

Received: 9 July 2022 Accepted: 26 July 2022 Published: 28 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

While the above-mentioned advantages of mRNA and the progress in mRNA engineering technologies are encouraging the realization of its clinical applications, properties of the mRNA, including instability in an in vivo environment and cell membrane impermeability, prevent it from reaching the cytoplasm efficiently, where it functions. Therefore, it is essential to develop a technology to efficiently deliver mRNA to the cytoplasm of target cells. mRNA delivery systems include lipoplex and lipid nanoparticles (LNPs) [2,14–18]. A Comirnaty (Pfizer (New York, NY, USA)/BionTech (Mainz, Germany)) and Spikevax (Moderna (Cambrige, MA, USA)) are examples of LNP-based mRNA vaccines against COVID-19 [8].

LNPs are composed of pH-sensitive cationic lipids (CLs), phospholipids, cholesterol (Chol), and polyethylene glycol (PEG)-modified lipids. Chol contributes to the stability of LNPs [19,20]. Phospholipids mainly contribute to the formation and stabilization of the lipid membrane [21]. PEG lipids contribute to the regulation of particle size, dispersion stability, and blood retention [22–24]. pH-sensitive cationic lipids contain tertiary amino group(s) and are positively charged in a weakly acidic environment [25,26]. This is important for efficient mRNA encapsulation through electrostatic interactions. It is also important for delivering mRNA to the cytoplasm by escape via membrane fusion from acidified endosomes after the LNPs have been internalized into cells through endocytosis [27,28]. Therefore, the lipid composition of the LNPs has significant effects on physicochemical properties as well as potency for drug delivery and, therefore, should be carefully optimized for each specific application and payload due to differences in the properties needed for the specific targeting of cell types or applications [29–31].

Dendritic cells (DCs) are able to initiate antigen-specific immune responses in lymphoid tissues after infectious pathogens are sensed and are among the most powerful antigen-presenting cells (APCs) [32,33]. Therefore, DCs are attractive APCs to be targeted for vaccination. However, the systemic delivery of mRNA to DCs faces a number of technical challenges. Although an ex vivo pulsed DC vaccine would have great potential because of its specificity, flexibility, and efficiency [34,35], it requires a complicated process that involves collecting a sample from the patient to re-administer to the patient. The direct targeting of DCs in vivo would be a good strategy for overcoming that drawback. Several studies have reported on DC targeting by modifying ligand molecules on the surface of the nanoparticles [36,37]. While this approach is widely used to achieve specific targeting, the formulation process is very complex and often poses challenges in terms of cost, reproducibility, and difficulty in characterization. On the other hand, Kranz, et al. reported on an RNA-lipoplex (LPX) that was prepared by mixing mRNA and ligand-unmodified liposomes composed of 1,2-di-*O*-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) [2]. Focusing on charge balance, specific gene expression in the spleen was achieved by designing a formulation with a negative charge ratio, which resulted in inducing a relatively selective gene expression in DCs. Antigen-specific antitumor immunity was induced in several cancer models by using an mRNA encoding a cancer antigen. Currently, BioNTech is conducting phase II clinical trials on an mRNA cancer vaccine formulation containing a fixed set of cancer-associated antigens (referred to as the FixVac platform) against advanced melanomas (ClinicalTrials.gov Identifier: NCT04526899) and human papillomavirus 16-positive head and neck squamous cell carcinomas (AHEAD-MERIT study, ClinicalTrials.gov Identifier: NCT04534205) as of the writing this manuscript. However, there are still few reports of LNPs that enable efficient mRNA delivery to DCs after systemic administration, and there is clearly substantial room for improvement regarding efficiency. In this study, we optimized mRNA-loaded LNPs targeting DCs using our originally developed CLs and the recently reported broad particle size control technology of LNPs synthesized using a microfluidic device [38,39] and compared the optimized LNPs with clinically-relevant LNP formulations.
