Polymeric nanoparticles for pulmonary protein and DNA delivery

Abstract

Polymeric nanoparticles (NPs) are promising carriers of biological agents to the lung due to advantages including biocompatibility, ease of surface modification, localized action and reduced systemic toxicity. However, there have been no studies extensively characterizing and comparing the behavior of polymeric NPs for pulmonary protein/DNA delivery both in vitro and in vitro. We screened six polymeric NPs: gelatin, chitosan, alginate, poly(lactic-co-glycolic) acid (PLGA), PLGA-chitosan and PLGA-poly(ethylene glycol) (PEG), for inhalational protein/DNA delivery. All NPs except PLGA-PEG and alginate were <300nm in size with a bi-phasic core compound release profile. Gelatin, PLGA NPs and PLGA-PEG NPs remained stable in deionized water, serum, saline and simulated lung fluid (Gamble's solution) over 5days. PLGA-based NPs and natural polymer NPs exhibited the highest cytocompatibility and dose-dependent in vitro uptake, respectively, by human alveolar type-1 epithelial cells. Based on these profiles, gelatin and PLGA NPs were used to encapsulate plasmid DNA encoding yellow fluorescent protein (YFP) or rhodamine-conjugated erythropoietin (EPO) for inhalational delivery to rats. Following a single inhalation, widespread pulmonary EPO distribution persisted for up to 10days while increasing YFP expression was observed for at least 7days for both NPs. The overall results support both PLGA and gelatin NPs as promising carriers for pulmonary protein/DNA delivery.

Keywords: DNA; Nanoparticles; Nebulization; Protein; Pulmonary.

Figure 1

TEM images of nanoparticles prepared using (A) gelatin, (B) chitosan, (C) alginate, (D) PLGA, (E) PLGA-CS and (F) PLGA-PEG. The insets represent the morphology of a single nanoparticle of each type.

Figure 2

Stability of all NPs was tested by measuring particle size in (A) DI water, (B) 10% FBS (C) saline solution and (D) simulated lung fluid at 37°C. The PLGA-based and gelatin NPs remained stable for up to 5 days while alginate NPs tended to show aggregation by the 4^th day. Chitosan NPs showed fluctuations in size indicating comparatively less stability.

Figure 3

Drug release studies done on all NPs using BSA as a protein model for a period of 21 days. All NPs showed a bi-phasic release consisting of a burst release for the first 2 days followed by sustained release for 3 weeks. Gelatin, Chitosan and PLGA NPs showed an initial burst release of more than 40% loaded protein within 4 days.

Figure 4

Type 1 alveolar epithelial cell viability studies using (A,B) MTS assay and (C,D) Picogreen ds DNA assay indicated that gelatin, chitosan, alginate and PLGA-PEG NPs maintained were cytocompatible up to a concentration of 1000 µg/ml. All NPs except alginate showed greater than 80% DNA content at 2000 µg/ml concentration. (n=3, *p<0.05 w.r.t control).

Figure 5

Cellular uptake of all six NPs by Type 1 alveolar epithelial cells was studied using Picogreen dsDNA assay and fluorescence readings following 2h incubation with NPs of increasing concentration. Results showed dose-dependence in uptake with increasing NP concentration (n=3, *p<0.05 w.r.t to cellular uptake at 100 µg/ml).

Figure 6

Panel A: Biofluorescence of rat lung slices fixed at 3, 5 and 7 d following nebulization of gelatin or PLGA based NPs loaded with YFP cDNA, compared to control lungs following nebulization of the corresponding NPs loaded with empty vector (bar=0.5 cm). The panels show increasing YFP expression up to 7 d following nebulization; expression was greater and more uniform using PLGA than gelatin NPs. Panel B: Confocal fluorescence microscopy of histological sections taken from the corresponding lungs shows increasing and widespread YFP expression up to 7 d post-inhalation compared to the respective controls (bar=50 µm).

Figure 7

Panel A: Biofluorescence of rat lung slices fixed at 1, 4, 7 and 10 d following nebulization of gelatin or PLGA-based NPs loaded with rhodamine-conjugated recombinant human erythropoietin (EPO-Rhodamine) compared to control lungs following nebulization of the corresponding empty NPs (bar=0.5 cm). Panel B: Confocal fluorescence microscopy of histological sections taken from the corresponding lungs. These panels show more sustained fluorescence up to 10 days post-inhalation using PLGA rather than gelatin NPs (bar=50 µm).