Encapsulation of alpha-1 antitrypsin in PLGA nanoparticles: in vitro characterization as an effective aerosol formulation in pulmonary diseases

Abstract

Background: Alpha 1-antitrypsin (α1AT) belongs to the superfamily of serpins and inhibits different proteases. α1AT protects the lung from cellular inflammatory enzymes. In the absence of α1AT, the degradation of lung tissue results to pulmonary complications. The pulmonary route is a potent noninvasive route for systemic and local delivery. The aerosolized α1AT not only affects locally its main site of action but also avoids remaining in circulation for a long period of time in peripheral blood. Poly (D, L lactide-co glycolide) (PLGA) is a biodegradable and biocompatible polymer approved for sustained controlled release of peptides and proteins. The aim of this work was to prepare a wide range of particle size as a carrier of protein-loaded nanoparticles to deposit in different parts of the respiratory system especially in the deep lung. Various lactide to glycolide ratio of the copolymer was used to obtain different release profile of the drug which covers extended and rapid drug release in one formulation.

Results: Nonaqueous and double emulsion techniques were applied for the synthesis of nanoparticles. Nanoparticles were characterized in terms of surface morphology, size distribution, powder X-ray diffraction (XRD), encapsulation efficiency, in vitro drug release, FTIR spectroscopy and differential scanning calorimetry (DSC). To evaluate the nanoparticles cytotoxicity, cell cytotoxicity test was carried out on the Cor L105 human epithelial lung cancer cell line. Nanoparticles were spherical with an average size in the range of 100 nm to 1μ. The encapsulation efficiency was found to be higher when the double emulsion technique was applied. XRD and DSC results indicated that α1AT encapsulated in the nanoparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The lactic acid to glycolic acid ratio affects the release profile of α1AT. Hence, PLGA with a 50:50 ratios exhibited the ability to release %60 of the drug within 8, but the polymer with a ratio of 75:25 had a continuous and longer release profile. Cytotoxicity studies showed that nanoparticles do not affect cell growth and were not toxic to cells.

Conclusion: In summary, α1AT-loaded nanoparticles may be considered as a novel formulation for efficient treatment of many pulmonary diseases.

Figure 1

Typical SEM image of AAT-loaded (a, b, c, d, f, g and h) and unloaded nanoparticles (e).

Figure 2

mean particle size of nanoparticle prepared with nonaqueous (a), double emulsion technique using PLGA 50:50 (b) and 75:25 (c).

Figure 3

XRD spectra of PLGA 50:50 (a), PLGA 75:25 (b), AAT- loaded PLGA nanoparticle prepared using nonaqueous emulsion technique (c), AAT- loaded nanoparticle prepared using double emulsion technique containing PLGA 50:50 (d) and AAT- loaded nanoparticle prepared using double emulsion technique containing PLGA 75:25 (e).

Figure 4

In vitro release profile of α1AT from PLGA nanoparticles with different ratio synthesized by nonaqueous and double emulsion technique.

Figure 5

FTIR spectra of pure AAT (a), PLGA 50:50 (b), PLGA 75:25 (c), nonaqueous emulsion made α1AT-loaded PLGA (d), double emulsion made α1AT-loaded PLGA (50:50) and (75:25)(e and f, respectively).

Figure 6

DSC thermograms PLGA, and α1AT -loaded PLGA nanoparticles: (a) PLGA 50:50; (b) PLGA 75:25; (c) α1AT -loaded nanoparticles prepared by nonaqueous emulsion method; (d) α1AT -loaded nanoparticles prepared by double emulsion method.

Figure 7

Cor L105 lung epithelial-like cells before and after treatment with nanoparticles.

Figure 8

a schematic representation of nanoparticle passage and decomposition in lung.

Figure 9

Two different methods of nanoparticle preparation by a) double emulsion and b) nonaqueous emulsion technique.