Interaction of bronchoalveolar lavage fluid with polyplexes and lipoplexes: analysing the role of proteins and glycoproteins.
BACKGROUND
Plasmid DNA complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has been used for gene transfer into the lung. Topical gene administration of lipoplexes or polyplexes into the lung after intratracheal instillation or aerosolisation could cause interaction of the complexes with extracellular substances of the airway surface liquid (ASL). These extracellular interactions might be causal for the observed inefficient transfection rate in vivo after topical administration. Therefore, we studied the impact of bronchoalveolar lavage fluid (BALF) on reporter gene expression mediated by non-viral gene vectors. BALF was considered as a model system to mimic possible interactions of the gene vectors with the ASL.


METHODS
BALF was taken from 15 patients who underwent diagnostic bronchoscopy. Lipoplexes and polyplexes were incubated with increasing concentrations of BALF and major components of the BALF such as albumin, mucin and alpha(1)-glycoprotein, as a representative of glycosylated proteins. As cationic polymers, we tested dendrimers (fractured PAMAM) and polyethylenimine 25 kDa (PEI) and, as cationic liposomes, we used Lipofect-AMINE. The effect of BALF on polyplexes and lipoplexes was analysed by transfection experiments, fluorescence-quenching assay, 2-D-gel electrophoresis, SDS-PAGE, DNAse protection assay, size and zeta-potential measurements.


RESULTS
BALF inhibited polyplex- and lipoplex-mediated gene transfer. Analysing components of BALF, we found that dendrimer-mediated gene transfer was not inhibited by any specific component. PEI-mediated gene transfer was dose-dependently inhibited by alpha(1)-glycoprotein, slightly inhibited by mucin, but not inhibited in the presence of albumin. Lipoplex-mediated gene transfer was inhibited by mucin at higher concentrations and by albumin, but not by alpha(1)-glycoprotein. 2-D-gel electrophoresis revealed that proteins of the BALF were adsorbed more intensively to lipoplexes than to polyplexes. In addition, mucin and alpha(1)-glycoprotein also adsorbed more intensively to lipoplexes than to polyplexes. Adsorption of BALF components led to a decrease in the positive zeta-potential of lipoplexes and led to a negative zeta-potential of polyplexes. Complement cleavage fragment C3 beta, and in the case of lipoplexes also the C3 alpha fragment, were found among the proteins opsonised on gene vectors.


CONCLUSIONS
Our study shows that BALF contains inhibitory components for non-viral gene transfer. We could not detect a specific inhibitory component, but inhibition was most likely due to the change in the surface charge of the gene vectors. Interestingly, there is evidence for complement activation when the route of pulmonary gene vector administration is chosen. Consequently, shielding of gene vectors to circumvent interaction with the ASL environment should be a focus for pulmonary administration in the future.
Rosenecker
J
J
Division of Molecular Pulmonology, Department of Pediatrics, Ludwig-Maximilians-Universität Munich, Germany. joseph.rosenecker@kk-i.med.uni-muenchen.de
Naundorf
S
S
Gersting
S W
SW
Hauck
R W
RW
Gessner
A
A
Nicklaus
P
P
Müller
R H
RH
Rudolph
C
C
D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans
D006023:Glycoproteins