. 2015 Aug 26;7(3):233-54.

doi: 10.3390/pharmaceutics7030233.

Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying

Tomoyuki Okuda¹, Yumiko Suzuki², Yuko Kobayashi³, Takehiko Ishii⁴, Satoshi Uchida⁵, Keiji Itaka⁶, Kazunori Kataoka^{7

8}, Hirokazu Okamoto⁹

Affiliations

¹ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. tokuda@meijo-u.ac.jp.
² Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. 124331505@ccalumni.meijo-u.ac.jp.
³ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. g0673221@ccalumni.meijo-u.ac.jp.
⁴ Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8656, Japan. tishii@bmw.t.u-tokyo.ac.jp.
⁵ Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. suchida@bmw.t.u-tokyo.ac.jp.
⁶ Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. itaka-ort@umin.net.
⁷ Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8656, Japan. kataoka@bmw.t.u-tokyo.ac.jo.
⁸ Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. kataoka@bmw.t.u-tokyo.ac.jo.
⁹ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. okamotoh@meijo-u.ac.jp.

PMID: 26343708
PMCID: PMC4588198
DOI: 10.3390/pharmaceutics7030233

Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying

Tomoyuki Okuda et al. Pharmaceutics. 2015.

. 2015 Aug 26;7(3):233-54.

doi: 10.3390/pharmaceutics7030233.

Authors

Tomoyuki Okuda¹, Yumiko Suzuki², Yuko Kobayashi³, Takehiko Ishii⁴, Satoshi Uchida⁵, Keiji Itaka⁶, Kazunori Kataoka^{7

8}, Hirokazu Okamoto⁹

Affiliations

¹ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. tokuda@meijo-u.ac.jp.
² Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. 124331505@ccalumni.meijo-u.ac.jp.
³ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. g0673221@ccalumni.meijo-u.ac.jp.
⁴ Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8656, Japan. tishii@bmw.t.u-tokyo.ac.jp.
⁵ Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. suchida@bmw.t.u-tokyo.ac.jp.
⁶ Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. itaka-ort@umin.net.
⁷ Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8656, Japan. kataoka@bmw.t.u-tokyo.ac.jo.
⁸ Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. kataoka@bmw.t.u-tokyo.ac.jo.
⁹ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. okamotoh@meijo-u.ac.jp.

PMID: 26343708
PMCID: PMC4588198
DOI: 10.3390/pharmaceutics7030233

Abstract

In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-μm diameter, and the integrity of plasmid DNA could be maintained during powder production. Furthermore, it was clarified that PEG-PAsp(DET)-based dry gene powder could more sufficiently maintain both the physicochemical properties and in vitro gene transfection efficiencies of polyplexes reconstituted after powder production than PAsp(DET)-based dry gene powder. From an in vitro inhalation study using an Andersen cascade impactor, it was demonstrated that the addition of l-leucine could markedly improve the inhalation performance of dry powders prepared by SFD. Following pulmonary delivery to mice, both PAsp(DET)- and PEG-PAsp(DET)-based dry gene powders could achieve higher gene transfection efficiencies in the lungs compared with a chitosan-based dry gene powder previously reported by us.

Keywords: biodegradable polycations; dry powder inhalers (DPIs); porous particles; pulmonary gene transfection; spray freeze drying (SFD).

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Figures

**Figure 1**
Chemical structures of (A) PAsp(DET) and (B) PEG-PAsp(DET).

**Figure 3**
Scanning electron micrographs of (A) Man DP, (B) Man-Leu DP, (C) PAsp(DET) DP (N/P = 4), and (D) PEG-PAsp(DET) DP (N/P = 40).

**Figure 4**
Integrity of pDNA in PAsp(DET) DP and PEG-PAsp(DET) DP after powder production by SFD. (A,H) naked pDNA, (B,G) pDNA digested with Hind III, (C,D) PAsp(DET) polyplex before and after powder production of PAsp(DET) DP (N/P = 4), (E,F) PAsp(DET) polyplex before and after powder production of PAsp(DET) DP (N/P = 8), (I,J) PEG-PAsp(DET) polyplex before and after powder production of PEG-PAsp(DET) DP (N/P = 40), and (K,L) PEG-PAsp(DET) polyplex before and after powder production of PEG-PAsp(DET) DP (N/P = 80). Electrophoresis of the samples for PAsp(DET) DP and PEG-PAsp(DET) DP was separately carried out with the individual gels. In the fluorescent image of each gel, the regions related to this study (shown as solid and dotted lines) were selectively cut off to combine with no modification. The position of each band in the combined images is the same as that in the original ones.

**Figure 5**
(A) Particle size and (B) zeta potential of polyplexes formed before and after powder production by SFD. Each powder was dissolved in ultra-pure water to reconstitute the polyplex. Each value represents the mean ± S.D. (n = 3). Significant difference compared with before power production (**, P < 0.01).

**Figure 6**
*In vitro* gene transfection efficiencies against CT26 cells by polyplexes formed before and after powder production by SFD. Each powder was dissolved in ultra-pure water to reconstitute the polyplex. Each value represents the mean ± S.D. (n = 4). Significant difference compared with before powder production (**, P < 0.01; *, P < 0.05).

**Figure 7**
*In vitro* deposition patterns of Man DP, Man-Leu DP, PAsp(DET) DP (N/P = 4), and PEG-PAsp(DET) DP (N/P = 40) in an 8-stage Andersen cascade impactor following inspiration at a flow rate of 28.3 L/min and for a flow time of 5 s. Each value represents the mean ± S.D. (n = 3).

**Figure 8**
Optical images of pulmonary delivery and gene expression by (A) PAsp(DET) DP (N/P = 4), (B) PEG-PAsp(DET) DP (N/P = 40), (C) PAsp(DET) SL (N/P = 4), (D) PEG-PAsp(DET) SL (N/P = 40), and (E) Chitosan DP (N/P = 10). The pulmonary delivery and gene expression were evaluated by the detection of fluorescence derived from ICG (Fl) and luminescence corresponding to luciferase activity (Lu) using IVIS^® following pulmonary administration to mice, respectively. The color scales are in photons/s/cm²/sr.

**Figure 9**
Correlation between pulmonary delivery and gene expression by PAsp(DET) DP and SL (N/P = 4), PEG-PAsp(DET) DP and SL (N/P = 40), and Chitosan DP. In the correlation diagram, fluorescence intensity derived from ICG at 15 min and maximum luminescence intensity corresponding to luciferase activity, which were calculated by IVIS^®, following pulmonary administration into mice were plotted, respectively. The calculated intensities were corrected using each pre-administered mouse.

**Figure 10**
Histological observation of lungs excised at 48 h following the pulmonary delivery of (C) PAsp(DET) DP (N/P = 4) and (D) PEG-PAsp(DET) DP (N/P = 40) into mice. (A) Water and (B) Man-Leu DP were administered as negative controls, and (E) lipopolysaccharide (120 mg/kg) was a positive control.

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Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying

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Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying

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