**5. Patents**

K. Sasaki, Y. Sato, and H. Harashima have filed intellectual property patents related to this publication.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14081572/s1, Synthesis of CL7H6, Figure S1: Reproducibility of physicochemical properties of the 14 LNPs (A-1 to A-14) examined in screening A, Figure S2: Statistical information for the effective design-based model selection for definitive screening design to predict the ζ-average of the LNPs (A-1 to A-14), Figure S3: Statistical information for the effective design-based model selection for definitive screening design to predict the percentage of RNA encapsulation of the LNPs (A-1 to A-14), Figure S4: Reproducibility of outputs of in vivo experiments in screening A, Figure S5: Correlation between ζ-average and cellular uptake or Nluc

expression in splenic DCs, Figure S6: Correlation between ζ-average and Nluc expression, Figure S7: I-A/I-E expression in splenic DCs after intravenous injection of LNPs examined in screening A and B, Figure S8: Stability of the A-11-LNPs, Figure S9: Examination of dose of the OVA mRNA-loaded A-11-LNPs for therapeutic antitumor experiment, Figure S10: Serum IFNβ level after an intravenous injection of OVA mRNA-loaded formulations at a dose of 0.03 mg mRNA/kg, Figure S11: Therapeutic antitumor effect of the A-11-LNPs on high dose conditions, Figure S12: Examination of formation of memory cells, Table S1: Full data of outputs of in vivo experiment in screening A, Table S2: Formulation conditions and physicochemical properties of the LNPs examined in screening B, Table S3: Full data of outputs of in vivo experiment in screening B.

**Author Contributions:** Conceptualization, Y.S. and H.H.; methodology, Y.S. and H.H.; validation, Y.S. and H.H.; formal analysis, Y.S.; investigation, K.S., K.O. and K.I.; resources, Y.S.; data curation, K.S., Y.S., K.O. and K.I.; writing—original draft preparation, K.S., Y.S., K.O. and K.I.; writing—review and editing, K.S., Y.S., K.O., K.I. and H.H.; visualization, Y.S.; supervision, Y.S. and H.H.; project administration, Y.S. and H.H.; funding acquisition, Y.S. and H.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported, in part, by the Hokkaido University Support Program for Frontier Research, The Mochida Memorial Foundation for Medical and Pharmaceutical Research and Research Expenses from the Ministry of Education, Culture, Sports, Science and Technology.

**Institutional Review Board Statement:** The experimental protocols were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the guidelines for the care and use of laboratory animals (approval number: 16-0015, date 28 March 2016; 20-0176, date 1 March 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors wish to express their gratitude to both Manabu Tokeshi and Masatoshi Maeki (Division of Applied Chemistry, Faculty of Engineering, Hokkaido University) for providing a microfluidic device (iLiNP device). The authors also wish to thank Milton S. Feather for his helpful advice in preparing the English manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
