Molecular Therapy - Nucleic Acids Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo  Krista Freimann, Piret Arukuusk, Kaido Kurrikoff, Ly Pärnaste, Raivo Raid, Andres Piirsoo, Margus Pooga, Ülo Langel  Molecular Therapy - Nucleic Acids  Volume 10, Pages 28-35 (March 2018) DOI: 10.1016/j.omtn.2017.10.011 Copyright © 2017 The Authors Terms and Conditions

Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10. 1016/j. omtn Copyright © 2017 The Authors Terms and Conditions

Figure 1 Size Distribution of Different Formulations of NF55/DNA Nanoparticles Size distribution of NF55/DNA nanoparticles in formulation 1 (green line, peptide concentrations 40 μM for in vitro studies) and formulation 2 (dashed red line, 400 μM for in vivo studies). Data were analyzed using the Malvern Zetasizer. Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10.1016/j.omtn.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Size Distribution of NF55/DNA Nanoparticles after the Filtration of Formulation 2 Particles The dashed red line represents the size distribution of NF55/DNA particles without filtration, and the green lines illustrate nanoparticles after filtration through the 200-nm filter. The dotted darker green line shows the size distribution of filtered nanoparticles after incubation for 60 min at room temperature. Data were analyzed using the Malvern Zetasizer. Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10.1016/j.omtn.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Transfection Efficacy of Filtered NF55/pDNA Nanoparticles Transfection efficacy of filtered NF55/pDNA nanoparticles in CHO cells at CR4 (A) and in BALB/c mice at CR4 (B). Light gray bars (Filtered) represent filtration results; dark gray bars (Filtered, dose-matched) represent the filtered nanoparticles with matched dose to account for the loss of material during the filtration. Untreated cells (UT) were used as a negative control, and Lipofectamine 2000 (LF2000) was used as a positive control. The data are presented as the mean ± SEM (n = 5). Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10.1016/j.omtn.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Size Distribution of CCC NF55/DNA Nanoparticles The particles obtained by complex formulation method 2 are represented with a dashed red line, and the cryo-concentration complex (CCC) formulation strategy is illustrated with a green line. The dotted darker green line shows the size distribution of CCC particles after incubation for 60 min at room temperature. Data were analyzed using the Malvern Zetasizer. Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10.1016/j.omtn.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Morphology of NF55/pDNA Nanoparticles Formulated in Different Conditions The nanoparticles were visualized by negative staining with uranyl acetate and transmission electron microscopy. (A) Formulation 2, (B) filtered formulation 2, (C) cryo-concentrated and reconstituted formulation 1. Scale bars, 200 nm. Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10.1016/j.omtn.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Resistance of NF55/DNA Complexes to Proteinase K Treatment Resistance to Proteinase K treatment of NF55/DNA complexes formed according to standard formulation methods (formulation 1 and formulation 2) and cryo-concentration (CCC) formulation strategy. The results were normalized to total DNA amount in the complexes. Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10.1016/j.omtn.2017.10.011) Copyright © 2017 The Authors Terms and Conditions

Figure 7 Expression of Marker Gene in BALB/c Mice Transfected by NF55/pDNA Nanoparticles Produced by CCC and Standard Formulation 2 at CR4 The data from at least five representative experiments are presented as the mean ± SEM, *p < 0.05 (Student’s t test, two-tailed distribution, two-sample unequal variance). Molecular Therapy - Nucleic Acids 2018 10, 28-35DOI: (10.1016/j.omtn.2017.10.011) Copyright © 2017 The Authors Terms and Conditions