Minimal toxicity of stabilized compacted DNA nanoparticles in the murine lung

Abstract

Nanoparticles containing DNA compacted with poly-l-lysine modified on an N-terminal cysteine with polyethylene glycol can effectively transfect cells of the airway epithelium when applied by the luminal route. To evaluate the toxicity of these nanoparticles, we administered 10 and 100 microg DNA compacted into nanoparticles suspended in normal saline by the intranasal route to mice and determined the pulmonary and systemic responses to this challenge, compared to administration of saline alone, and in some experiments, compared to administration of naked DNA, Escherichia coli genomic DNA, or lipofectin-complexed naked DNA. There was no systemic response to either dose of nanoparticles in serum chemistries, hematologic parameters, serum complement, IL-6, or MIP-2 levels or in the activity, growth, and grooming of the mice. Nanoparticles containing 10 microg DNA induced responses comparable to saline in all measures, including BAL cell counts and differentials and cytokine levels and histology. However, mice dosed with 100 microg DNA in nanoparticles had modest increases in BAL neutrophils 48 and 72 h after dosing, modest increases in BAL IL-6 and KC beginning 24 and 48 h, respectively, after dosing, and, on histology of the lung, a trace to 1+ mononuclear cell infiltrates about the pulmonary veins at 48 h, which were markedly reduced by 10 days and gone by 28 days after dosing. BAL neutrophil and cytokine responses were no greater than those entrained by naked DNA for up to 24 h. However, compared to administration of only 10 microg E. coli genomic DNA, the response to compacted DNA was much less. A low dose of lipofectin-complexed DNA (5 microg DNA) induced the same response as 20-fold higher doses of DNA nanoparticles. These data indicate that DNA nanoparticles have no measurable toxic effect at a dose of 10 microg and a very modest effect, which is not limiting, at a dose of 100 microg, which gives maximal gene expression. This favorable toxicity profile encourages development of stabilized compacted DNA for airway administration.

Fatty acid–spermine conjugates as DNA carriers for nonviral in vivo gene delivery

“…[36][37][38] The most successful cases occur after intranasal or intratracheal administration, when targeting airway epithelial cells. 3,5,9,39 Tissues containing nondividing cells (as in muscle) or slow mitotic cells (as in the liver) are very attractive targets because pDNA can remain for a long time inside the cells. 40 In relation to the relatively fast kinetics seen here, we do not exclude the possibility that the decrease in expression after 5-7 days may be indicative of toxicity.…”

“…Compared with physical methods, chemical systems have low efficiency and, in spite of their safety, they frequently show toxic properties because of the high concentrations of carriers that are needed to obtain significant gene expression. [5][6][7][8] Thus, highly efficient nonviral gene carriers are needed. For this purpose, DNA condensation is a recurrent requisite for this type of nonviral therapeutic approach.…”

“…For this purpose, DNA condensation is a recurrent requisite for this type of nonviral therapeutic approach. 5,[8][9][10][11][12] Bloomfield 13 has defined DNA condensation as the collapse of extended DNA chains into compact, orderly particles containing only one or a few molecules. Typically, this process results from a neutralization of the negative charges of the DNA phosphate groups with transition into the ordered phase that occurs when 90% of charges are neutralized.…”

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung

“…In vivo gene transfer following aerosol delivery to the lung has been demonstrated previously with GL67A in both rodents and humans [12][13][14] and with PEI in rodents and sheep. 9,10,15,16 Evidence for in vivo gene transfer following administration of NP to the lungs of mice 17,18 and to CF patients has been reported 19 and although not delivered by aerosol in these studies, maintenance of the physical properties and biological function of NP collected post-aerosolisation has been described. 20 Many differences in the delivery protocols, the DNA vectors and the model systems have made it difficult to assess if any of these GTAs has a significant advantage over the others.…”

“…The vectors used in this study were all 'first generation' plasmids, which contain high numbers (4300) of CpG motifs, therefore, it is possible that this inflammatory response could be attributed, at least in part, to the presence of non-methylated CpG motifs in the bacterial pDNA, 28 especially, as the GL67A-treated sheep received the largest plasmid dose (52.8 mg in 20 ml). Although the plasmid dose received by the NP group was only B20% less, it has been argued that the markedly reduced inflammation in response to instillation of NP in the mouse lung is due to the pDNA within the NP formulation being masked from the toll-like receptor 9 in the endosome, 18 as a consequence of a nucleolin-mediated route of entry to the cell and into the nucleus. 29 Despite this, a modest BAL neutrophil response has been reported for a 100 mg dose administered to the lungs of Balb/c mice, 18 which could be consistent with the trend towards increased BAL neutrophils observed after lung delivery in the sheep.…”