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A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting.

Wang SM, He X, Li N, Yu F, Hu Y, Wang LS, Zhang P, Du YK, Du SS, Yin ZF, Wei YR, Mulet X, Coia G, Weng D, He JH, Wu M, Li HP - Int J Nanomedicine (2015)

Bottom Line: Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17.Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity.Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People's Republic of China.

ABSTRACT
Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies' potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

No MeSH data available.


Related in: MedlinePlus

Analysis of the binding specificity of Nb6 and Nb17 to rSPA.Notes: (A) Western blot showed that Nb6 (lane 1) and Nb17 (lane 2) bound to rSPA at 19 kDa, lane 3 are mark. (B) ELISA showed excellent binding activity of Nb6 (lane 1) and Nb17 (lane 2) to rSPA compared to the negative control (lane 3). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.
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f3-ijn-10-2857: Analysis of the binding specificity of Nb6 and Nb17 to rSPA.Notes: (A) Western blot showed that Nb6 (lane 1) and Nb17 (lane 2) bound to rSPA at 19 kDa, lane 3 are mark. (B) ELISA showed excellent binding activity of Nb6 (lane 1) and Nb17 (lane 2) to rSPA compared to the negative control (lane 3). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.

Mentions: Two clones, Nb6 and Nb17, were chosen for further characterization. Expression conditions of both sequences were analyzed using various temperatures and IPTG concentrations. Optimal expression levels were attained at 25°C for 4 hours with 0.8 mM IPTG. The molecular weights of these proteins were approximately 19 kDa and were soluble. The concentration of the purified proteins was approximately 25 mg/mL. With immunoblotting, rSPA reacted well with purified Nb6 and Nb17 with a single band of 19 kDa (Figure 3A). ELISA also showed that the purified Nb6 and Nb17 had positive reactivity to rSPA (Figure 3B).


A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting.

Wang SM, He X, Li N, Yu F, Hu Y, Wang LS, Zhang P, Du YK, Du SS, Yin ZF, Wei YR, Mulet X, Coia G, Weng D, He JH, Wu M, Li HP - Int J Nanomedicine (2015)

Analysis of the binding specificity of Nb6 and Nb17 to rSPA.Notes: (A) Western blot showed that Nb6 (lane 1) and Nb17 (lane 2) bound to rSPA at 19 kDa, lane 3 are mark. (B) ELISA showed excellent binding activity of Nb6 (lane 1) and Nb17 (lane 2) to rSPA compared to the negative control (lane 3). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403696&req=5

f3-ijn-10-2857: Analysis of the binding specificity of Nb6 and Nb17 to rSPA.Notes: (A) Western blot showed that Nb6 (lane 1) and Nb17 (lane 2) bound to rSPA at 19 kDa, lane 3 are mark. (B) ELISA showed excellent binding activity of Nb6 (lane 1) and Nb17 (lane 2) to rSPA compared to the negative control (lane 3). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.
Mentions: Two clones, Nb6 and Nb17, were chosen for further characterization. Expression conditions of both sequences were analyzed using various temperatures and IPTG concentrations. Optimal expression levels were attained at 25°C for 4 hours with 0.8 mM IPTG. The molecular weights of these proteins were approximately 19 kDa and were soluble. The concentration of the purified proteins was approximately 25 mg/mL. With immunoblotting, rSPA reacted well with purified Nb6 and Nb17 with a single band of 19 kDa (Figure 3A). ELISA also showed that the purified Nb6 and Nb17 had positive reactivity to rSPA (Figure 3B).

Bottom Line: Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17.Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity.Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People's Republic of China.

ABSTRACT
Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies' potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

No MeSH data available.


Related in: MedlinePlus