Intratracheally instilled mannosylated cationic liposome/NFκB decoy complexes for effective prevention of LPS-induced lung inflammation
Graphical abstract
Introduction
Pulmonary diseases such as chronic obstructive pulmonary disease [1], acute lung injury [2], and pulmonary allergy [3] are the three leading causes of morbidity, mortality and disease-related decreases in quality of life worldwide [4]. The fundamental pathogenesis of these diseases involves the progression of abnormal inflammatory responses against stimuli in the lung. The production of inflammatory mediators including cytokines such as TNF-α and IL-1β, chemokines such as CINC-1, and adhesion molecules are an initial response mediated by resident macrophages and mast cells inciting neutrophil infiltration for facilitation of inflammatory tissue damage [5]. Nuclear factor kappa B (NFκB) is a key transcription factor that regulates the expression of multiple genes involved in inflammation [6], [7]. Following activation, NFκB, which is primarily a heterodimer of p50 and p65 subunits and sequestered in the cytosol, is unmasked from the inhibitory protein IκBα and then translocates to the nucleus where it binds to a consensus binding site for transcription of a number of inflammatory genes. Recently, decoy strategies have lead a revolution in the treatment of inflammation [8], [9], [10] due to their promise of high specificity to target genes and low toxicity in comparison to conventional steroid therapy [11]. A double-stranded oligonucleotide (ODN) NFκB decoy corresponding to the cis-NFκB binding sequence, is a competitive ODN of NFκB for endogenous cis-NFκB binding elements with subsequent modulation of NFκB-dependent gene expression [12], [13]. Therefore, a specific inhibition of activated NFκB by NFκB decoy is a new anti-inflammatory therapeutic approach.
A large body of evidence has strongly demonstrated a clear role of resident alveolar cells, especially alveolar macrophages in lung inflammation [14], [15]. Matsuda and colleagues [16] reported the intravenous injection of NFκB decoy for an effective treatment of sepsis-induced acute lung injury. Intravenously injected NFκB decoy is rapidly eliminated from blood circulation with high renal clearance due to its anionic charge, small size (13.2 kDa) and enzymatic degradation. Consequently, there are very low levels of NFκB decoy deposition in the lungs [17]. This evidence suggests that a high dose of NFκB decoy (500 μg/mouse) is required for penetration into the airway epithelial cells and macrophages after intravenous administration [18]. Therefore, direct pulmonary delivery by inhalation may increase lung deposition of NFκB decoy for improved therapeutic efficacy [19], [20]. A limitation of inhaled NFκB decoy is the lack of ability to efficiently target alveolar macrophages, resulting in an insufficient treatment of lung inflammation [21]. These results emphasize the necessity of developing a system to target alveolar macrophages with NFκB decoy for anti-inflammatory therapy.
Alveolar macrophage-selective targeting using sugar-modified nanoparticles is of great interest since mannose receptors on the cell surface can recognize mannose and fucose moieties [22]. We previously developed mannosylated (Man) and fucosylated (Fuc) liposomes for liver macrophage-selective uptake via mannose-receptor mediated endocytosis after intravenous administration [23]. Recently, we have reported the effective alveolar macrophage-selective targeting of Man-liposomes via mannose receptor-mediated endocytosis after intratracheal administration [24] for improved therapeutic effects [25]. These observations correspond to a study by Chono and colleagues who reported that intratracheally instilled Man-liposomes are taken up by alveolar macrophages in a size-dependent manner via mannose receptor-mediated mechanism [26]. As far as a carrier of NFκB decoys is concerned, the cationic nature of Man-liposomes is necessary for complex formation via electrostatic interaction. We could not, however, ensure the effective targeting of Man-cationic liposome/NFκB decoy complexes to alveolar macrophages.
We evaluated the targeting efficiency of Man-cationic liposome/NFκB decoy complexes by means of lung deposition and cellular localization of NFκB decoy in alveolar macrophages after intratracheal administration in rats. Their therapeutic potentials including inhibition of cytokine and chemokine release, neutrophil infiltration as well as neutrophilic myeloperoxidase (MPO) activity and NFκB activation were investigated in a LPS-induced lung inflammation model.
Section snippets
Materials
Decoy ODN are phosphorothioate double-stranded DNA and were kindly provided by Anges MG Inc. (Osaka, Japan). The 20mer-NFκB decoys containing the consensual NFκB binding sites were 5′-GGAGGGAAATCCCTTCAAGG-3′ (strand A) and 3′-CCTCCCTTTAGGGAAGTTCC-5′ (strand B). The scrambled decoys, non-sense sequences to NFκB were 5′-TTGCCGTACCTGACTTAGCC-3′ (strand A) and 3′-AACGGCATGGACTGAATCGG-5′ (strand B). Control CpG ODN 1668, was purchased from Invitrogen (San Diego, CA). LPS 0111:B4, MPO enzyme and o
Results and discussion
To develop an effective decoy strategy that can be translated into a clinical setting for lung inflammatory treatment, several factors have to be considered. These include choice of therapeutic NFκB decoy, alveolar macrophage-targeting vectors and route of administration. We optimized Man-cationic liposome/NFκB decoy complexes for alveolar macrophage-selective targeting and demonstrated a sufficient inhibition of LPS-induced lung inflammation after intratracheal administration of these
Conclusion
We have successfully demonstrated high levels of deposition in the lung with alveolar macrophage-selective localization of a NFκB decoy using Man-cationic liposomes via intratracheal administration. Man-cationic liposome/NFκB decoy complexes significantly inhibited NFκB and downstream inflammatory mediators after intratracheal administration in a LPS-induced lung inflammation model. This study underlines a key role of Man-cationic liposomes as effective targeting systems, and proves the concept
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by the Japan Society for the Promotion of Sciences (JSPS) through a JSPS Research Fellowship for Young Scientists.
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