Aqueous environments in living cells are crowded, with up to >50 wt% small and macromolecule-size solutes. We investigated quantitatively one important consequence of molecular crowding--reduced diffusion of biologically important solutes. Fluorescence correlation spectroscopy (FCS) was used to measure the diffusion of a series of fluorescent small solutes and macromolecules. In water, diffusion coefficients (D(o)w) were (in cm2/s x 10(-8)): rhodamine green (270), albumin (52), dextrans (75, 10 kDa; 10, 500 kDa), double-stranded DNAs (96, 20 bp; 10, 1 kb; 3.4, 4.5 kb) and polystyrene nanospheres (5.4, 20 nm diameter; 2.3, 100 nm). Aqueous-phase diffusion (Dw) in solutions crowded with Ficoll-70 (0-60 wt%) was reduced by up to 650-fold in an exponential manner: Dw = D(o)w exp (-[C]/[C]exp), where [C]exp is the concentration (in wt%) of crowding agent reducing D(o)w by 63%. FCS data for all solutes and Ficoll-70 concentrations fitted well to a model of single-component, simple (non-anomalous) diffusion. Interestingly [C]exp were nearly identical (11+/-2 wt%, SD) for diffusion of the very different types of macromolecules in Ficoll-70 solutions. However, [C]exp was dependent on the nature of the crowding agent: for example, [C]exp for diffusion of rhodamine green was 30 wt% for glycerol and 16 wt% for 500 kDa dextran. Our results indicate that molecular crowding can greatly reduce aqueous-phase diffusion of biologically important macromolecules, and demonstrate a previously unrecognized insensitivity of crowding effects on the size and characteristics of the diffusing species.

The cytosol of mammalian cells is a crowded environment containing soluble proteins and a network of cytoskeletal filaments. Gene delivery by synthetic vectors involves the endocytosis of DNA-polycation complexes, escape from endosomes, and diffusion of non-complexed DNA through the cytosol to reach the nucleus. We found previously that the translational diffusion of large DNAs (>250 bp) in cytoplasm was greatly slowed compared with that of smaller DNAs (Lukacs, G. L., Haggie, P. Gene delivery by non-viral vectors is a complex and inefficient process that involves cellular internalization by endocytosis, escape from endosomes, DNA-polycation dissociation, and diffusion of non-complexed DNA in the cytoplasm to reach the nucleus (1-3). DNA diffusion in the cytoplasm and nuclear import are believed to be rate-limiting determinants of transgene delivery, extending the time that non-complexed DNA is exposed to cytosolic DNases. Remarkably, Ͻ0.1% of DNA microinjected into the cytosol is ultimately expressed (4, 5). We measured previously the mobility of differently sized DNAs in cytoplasm by photobleaching cells after microinjection with fluorescently labeled DNAs (6). The principal finding was that DNA diffusion in cytoplasm (D cyto ) was modestly slowed compared with that in saline (D o ) (D cyto /D o ϳ 0.2) for small DNA fragments of Ͻ250 bp but was greatly reduced (D cyto /D o Ͻ 0.05) for larger DNAs of Ͼ250 bp such as plasmid-sized DNAs. In contrast, the diffusion of dextrans and Ficolls, which are considered to be non-interacting macromolecules, was only mildly dependent on their size up to 500 -1000 kDa (7), which is equivalent in molecular mass to a 750 -1500-bp DNA fragment. Identification of the mechanism of reduced mobility of large DNAs in cytoplasm has important consequences regarding intrinsic limitations of non-viral gene delivery and in developing strategies to improve its efficacy.Several factors can in principle reduce the diffusion of a solute in cytoplasm versus saline, including fluid phase viscosity, binding, and crowding by mobile and immobile macromolecules (8). Systematic analysis of the diffusion of a small fluorescein-like molecule in cytoplasm indicated that reduced diffusion in cytoplasm (D cyto /D o ϳ 0.25) resulted mainly from macromolecular crowding by the relatively high concentration of proteins in cytoplasm of ϳ100 -150 mg/ml (reviewed in Refs. 9 and 10). Fluid phase viscosity, defined as the effective viscosity sensed by a small molecule that does not undergo binding or other macromolecular interactions, has little influence on cytoplasmic diffusion (11,12). Binding can greatly reduce apparent diffusion, as was found for some enzymes that assemble into macromolecular complexes (13). As a densely charged polyanion, DNA binding to cytoplasmic components could be an important factor in reducing its diffusion in cytoplasm as was found in the nucleus, where DNA is nearly immobile probably because of binding to positively charged histones (6,14). DNA diffusion in heterogeneous polydispers...

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C(12)-C(16)) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, xi-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes.