Cystic fibrosis (CF) is characterized by the presence of a viscoelastic mucus layer in the upper airways and bronchi. The underlying problem is a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein. Clinical studies of gene transfer for CF are ongoing. For gene delivery to the airways of CF patients to be effective, the mucus covering the target cells must be overcome. We therefore examined the extent to which CF sputum presents a physical barrier to the transport of nanospheres of a size comparable to that of lipoplexes and other transfection systems currently being clinically evaluated for CF gene therapy. We observed that an extremely low percentage of nanospheres (< 0.3%) moved through a 220-microm-thick CF sputum layer after 150 min. The largest nanospheres studied (560 nm) were almost completely blocked by the sputum, whereas the smaller nanospheres (124 nm) were retarded only by a factor of 1.3 as compared with buffer. Surprisingly, the nanospheres diffused significantly more easily through the more viscoelastic sputum samples. We hypothesize that the structure of the network in sputum becomes more macroporous when the sputum becomes more viscoelastic. Sputum from a patient with chronic obstructive pulmonary disease retarded the transport of nanospheres to the same extent as did CF sputum. When directly mixed with CF sputum, recombinant human deoxyribonuclease I moderately facilitated the transport of nanospheres through CF sputum.

Gene complexes with optimal physicochemical characteristics for cystic fibrosis (CF) gene therapy in vitro may become inactive in vivo as a result of destruction upon interaction with CF mucus. Therefore, we examined in this study to what extent main sputum components (linear DNA, mucin, phosphatidylcholine, phosphatidylglycerol, and albumin) may disintegrate lipoplexes. We found that mixing linear DNA with lipoplexes, in concentration ratios as occurs in the mucus of patients with CF in clinical studies with lipoplexes, drastically altered the surface charge and size of our lipoplexes and resulted in the liberation of plasmid DNA from the lipoplexes. These concentration ratios occur in vivo when the DNA concentration in the sputum becomes > 2.7 mg/ml, a quite realistic concentration even in patients without acute exacerbations. Lipoplexes brought in contact with native CF sputa at clinically relevant concentration ratios dissociated when the DNA concentration in the sputa was > 2.7 mg/ml. However, when the linear DNA was degraded by recombinant human deoxyribonuclease I before lipoplexes were added, the linear DNA did not cause any dissociation of the lipoplexes. Addition of albumin and mucin to the lipoplexes in a clinically relevant concentration ratio changed the surface charge of the lipoplexes to negative, however, without release of plasmid DNA. Mucin, dipalmitoylglycerophosphocholine, and dipalmitoylglycerophosphoglycerol did not cause any change in lipoplex properties at clinically relevant concentration ratios.

values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (⑀), as revealed from PCH analysis, was greater than ⑀ for the free RhONs, indicating that the Rh-ONs were attached to pDMAEMA chains.

In this study, dual-color fluorescence fluctuation spectroscopy (FFS) was explored to characterize the association of oligonucleotides (ONs) to cationic polymers. The results from dual-color FFS, in which both the ONs and the cationic polymers were fluorescently labeled, were compared with data obtained from single-color FFS in which only the ONs or the cationic polymers were fluorescently marked. As a model, the association of negatively charged 20-mer ONs to positively charged poly-Llysine (pLL) was studied. The binding of rhodamine green-labeled ONs (RhGr-ONs) to nonlabeled pLL could be clearly observed in the fluorescence fluctuation profiles. Especially, highly intense fluorescence peaks appeared in the fluorescence fluctuations. A highly intense fluorescence peak was considered to originate from one complex in which a number of RhGr-ONs were present. Complexing nonlabeled ONs to rhodamine green-labeled pLL (RhGr-pLL) also resulted in highly intense fluorescence peaks, indicating not only that the complexes consisted of a number of ONs but also that numerous RhGr-pLL chains were present. Upon complexation of red-labeled pLL (Cy5-pLL) to green-labeled ONs (RhGr-ONs), highly intense fluorescence peaks occurred simultaneously in the red and green detector. These data proved the multimolecular composition of the polyplexes and agreed with the observations from single-color FFS.

FCS seems applicable for study of the interactions between ONs and different types of cationic polymers.

With regard to the development of pharmaceutical carriers for oligonucleotides, the interactions between a cationic polymer, poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), and rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied. The composition of the pDMAEMA/Rh-ON complexes was investigated as a function of the charge ratio (φ; +/-) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. Gel electrophoresis revealed that at φ < 1 only a fraction of the short Rh-ONs bind the longer pDMAEMA chains. Dynamic light scattering and zeta potential (ζ) indicated that at φ < 1 only a fraction of the phosphate anions on the Rh-ONs are involved in complex formation which results in "dangling" of the Rh-ONs. Moreover, from fluorescence measurements, multimolecular complexes (i.e., several Rh-ONs per pDMAEMA chain) were revealed. At 1 < φ < 3, ζ approximated zero. This led to a clustering of individual complexes. At φ > 3, all the Rh-ONs were bound while, compared to complexes at φ < 1, the average number of Rh-ONs bound to one pDMAEMA chain was decreased. The presence of multimolecular complexes was confirmed from fluorescence correlation spectroscopy (FCS) measurements. The multimolecular complexes were observed as highly intense fluorescent particles upon their diffusion through the confocal volume of the FCS instrument. Also, the decrease in the average number of Rh-ONs bound to one pDMAEMA chain, upon increasing φ, was confirmed by FCS.

CF sputum drastically retards the diffusion of lipoplexes. DNA in the sputa aggregates the lipoplexes. This may lower the transport of lipoplexes through the sputa and gene expression. Pretreatment of CF patients with rhDNase I may enhance the efficiency of CF gene therapy, as it allows a better transport of the lipoplexes through the sputum and as it partly removes the sputum which will result in a thinner sputum layer on top of the epithelial cells.

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