The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H ؉ buffering polyamines by enhanced endosomal Cl ؊ accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl ؊ concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl ؊ ] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl ؊ or pH indicators. [Cl ؊ ] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl ؊ accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl ؊ accumulation and swelling/lysis were accounted for by the greater H ؉ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H ؉ /pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.Although gene delivery using non-viral vectors offers potential advantages over virus-based delivery systems, the relatively low transfection efficiency of non-viral vectors has been their major limitation for in vivo applications (1-4). The archetypal non-viral gene delivery system is the cationic polymer-DNA complex (polyplex), 1 in which plasmid DNA and a cationic carrier are condensed into a tight complex suitable for cellular internalization by endocytosis (5, 6). Transgene delivery to the nucleus is thought to require escape of the polyplex from endosomes, DNA/polymer dissociation, cytoplasmic DNA diffusion, and nuclear uptake (5, 7, 8). The low efficiency of polyplex escape from endosomes is thought to be an important determinant of the overall efficiency of non-viral gene transfer. Polyamines are useful polycationic macromolecules for nonviral gene transfer because of their high density of positive charges, ease of synthesis, and efficient polyplex formation (8 -10). Cationic polyamines with fixed, non-titratable charges such as polylysine (POL) are substantially less efficient at gene transfer than polyamines with titratable amines such as polyamidoamine (PAM) (11) and polyethylenimine (PEI) (12, 13). The lower efficiency is not caused by differences in morphology of the complexes or cell association (14). To explain this observation it has been postulated without direct evidence that the high H ϩ buffer capacity of polyamines containing titratable amines results in endosomal Cl Ϫ accumulation during acidification with presumed ...

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

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Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation, which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model, and near-complete suppression of cyst growth was found by tetrazolo-CFTR inh -172, a tetrazolo-derived thiazolidinone, and Ph-GlyH-101, a phenyl-derived glycine hydrazide, without an effect on cell proliferation. These compounds also inhibited cyst number and growth by Ͼ80% in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR inh -172 and Ph-GlyH-101 to neonatal, kidney-specific PKD1 knockout mice produced stable, therapeutic inhibitor concentrations of Ͼ3 M in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD.