Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA

Kuo, Jung-hua Steven; Hwang, Robin

doi:10.1211/0022357022494

Cited by 49 publications

(33 citation statements)

References 34 publications

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“…For detecting the electrophoretic bands corresponding to nucleic acids, polyanions such as polyaspartic acid and heparin are generally needed to unpack nucleic acids from nucleic acid/cationic vector complexes. 33,34) Without a polyanion in this study, however, the band corresponding to siRNA could be detected in the siRNA/chitosan complex reformed after dissolution of the siRNA/chitosan powder (Fig. 2), indicating the relatively weak electrostatic interaction between chitosan and siRNA compared with other cationic vectors such as polyethyleneimine.…”

Section: Discussionmentioning

confidence: 58%

Gene Silencing in a Mouse Lung Metastasis Model by an Inhalable Dry Small Interfering RNA Powder Prepared Using the Supercritical Carbon Dioxide Technique

Okuda

Kito

Oiwa

et al. 2013

Biological & Pharmaceutical Bulletin

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In this study, a novel dry small interfering RNA (siRNA) powder for inhalation, containing chitosan and mannitol, was prepared using the supercritical carbon dioxide (CO 2 ) technique. Although the siRNA/chitosan powder was difficult to disperse because of a long needle-like structure, it could be reduced to fragments of 10-20 µm by manual grinding, which allowed for administration into mice. Electrophoresis revealed that the supercritical CO 2 technique and manual grinding didn't greatly affect the integrity of the siRNA. Furthermore, the siRNA was more stable in the lungs than in blood, suggesting the utility of pulmonary delivery. Biodistribution experiments using Cy5.5-labeled siRNA demonstrated that pulmonary administration of the powder achieved a prolonged exposure of the siRNA/chitosan complex on the lung epithelial surface at a higher concentration. For the evaluation of the in-vivo gene silencing effect of the siRNA/chitosan powder, mice bearing colon26/Luc cells were used. The powder significantly inhibited the increase in luminescence intensity in the lungs, but the siRNA/chitosan solution and a non-specific dry siRNA/chitosan powder didn't, indicating the effective and specific gene silencing against the tumor cells metastasized in the lungs of mice by the siRNA/chitosan powder. These results strongly indicate that inhalable dry siRNA powders have the possibility of effective pulmonary gene silencing and that the supercritical CO 2 technique can be applied to the production.Key words small interfering RNA; inhalable dry powder; supercritical fluid technique; pulmonary gene delivery; chitosan RNA interference (RNAi), first reported by Fire et al., is a unique cellular phenomenon in which double-stranded RNA (dsRNA) regulates gene expression, contributing to cellular defenses against viral infections and transposon expansion.

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Section: Discussionmentioning

confidence: 58%

Gene Silencing in a Mouse Lung Metastasis Model by an Inhalable Dry Small Interfering RNA Powder Prepared Using the Supercritical Carbon Dioxide Technique

Okuda

Kito

Oiwa

et al. 2013

Biological & Pharmaceutical Bulletin

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“…157 They are usually low-density composite amorphous particles with high specific surface area. 158 Thermolabile protein and peptide substances, such as insulin 159 and plasmid DNA, 160 can also be formulated into dry powder inhalation products by SFD.…”

Section: Spray-freeze-drying (Sfd)mentioning

confidence: 99%

Nanomedicine in pulmonary delivery

Mansour¹,

Rhee²,

Wu³

2009

IJN

409

269

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“…Highly porous dry powders can be obtained by this method. 19,20) However, with the spray-freeze drying method, gene expression may be decreased due to the increase in particle size of the gene vectors during the process of freeze-drying. Many studies report that it is possible to maintain physical properties during freeze-drying.…”

Section: 7)mentioning

confidence: 99%

Bovine Serum Albumin as a Lyoprotectant for Preparation of DNA Dry Powder Formulations Using the Spray-Freeze Drying Method

Tsukamoto

Okuda

Okamoto

et al. 2012

Biological & Pharmaceutical Bulletin

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The development of efficient and selective therapeutic gene delivery methods is a potential medical treatment for intractable diseases. Dry powder inhalers (DPIs) can efficiently deliver drugs locally to the lung. Many reports discuss preparation methods for DPIs. Spray-freeze drying is a method by which highly porous particulates can be prepared. However, altered physical properties after preparation may result in changes in gene expression. In this study, bovine serum albumin (BSA) was added as a lyoprotectant, and 1,2-dioleoyl-3-trimethylammonium propane/cholesterol liposomes/pCMV-Luc DPIs (lipoplex DPIs) were prepared by spray-freeze drying. The mean particle sizes of the lipoplex DPIs prepared without BSA increased by approximately 6.7-fold compared with that of the lipoplexes solution. In contrast, the mean particle sizes of the lipoplex/BSA DPIs increased only slightly. Gene expression was evaluated after the intratracheal administration of the lipoplexes solution, with maximum gene expression observed at 12 h after the administration. In contrast, maximum gene expression of the lipoplex/BSA DPIs occurred at 6 h after administration. The gene expression associated with the lipoplex DPIs was significantly lower compared with that of the lipoplex/BSA DPIs at 6 (p<0.01), 12 (p<0.01), and 24 h (p<0.05). These variances may be due to the difference in mean particle size between the DPI formulations. The results suggest that BSA is a useful lyoprotectant for dry powder formulation preparations of DNA using the spray-freeze drying method, given that the preparation results in minimal variation of physical properties and gene expression.Key words dry powder inhaler; spray-freeze drying; lyoprotectant; bovine serum albumin; pulmonary gene delivery; lipoplex Gene therapy is expected to be a cure for intractable diseases. However, efficient and selective delivery of therapeutic genes to the target cells is necessary for gene therapy to be effective.1) Although viral vectors may show high gene expression, toxicity as a result of immunogenicity is a major problem associated with their use. Therefore, non-viral vectors have received attention because of their relatively low toxicity. 2)Many researchers have demonstrated that cationic liposomes show high gene expression and low toxicity.3-5) Lung diseases such as lung cancer and cystic fibrosis are intractable, and therefore, the realization of efficient gene therapy to treat such conditions is essential. However, the development of delivery methods that enable the local delivery of therapeutic genes to the lung is needed.The aerosolization of gene vectors is an efficient delivery method, and many related studies have been carried out. 6,7)However, it is necessary for aerosols to be sprayed immediately because the stability of gene vectors in aqueous solution is low. 8) Alternatively, dry powder inhalers (DPIs) are widely used and accepted as an effective system for pulmonary drug delivery. DPIs have many advantages, including increased shelf life of drugs including pla...

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Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA

Cited by 49 publications

References 34 publications

Gene Silencing in a Mouse Lung Metastasis Model by an Inhalable Dry Small Interfering RNA Powder Prepared Using the Supercritical Carbon Dioxide Technique

Gene Silencing in a Mouse Lung Metastasis Model by an Inhalable Dry Small Interfering RNA Powder Prepared Using the Supercritical Carbon Dioxide Technique

Nanomedicine in pulmonary delivery

Bovine Serum Albumin as a Lyoprotectant for Preparation of DNA Dry Powder Formulations Using the Spray-Freeze Drying Method

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