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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 20, 2004 as doi:10.1096/fj.03-0494fje. |
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Department of Gastroenterology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo;
* Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama;
First Department of Medicine, Osaka University School of Medicine, Suita, Osaka;
First Department of Internal Medicine, Akita University School of Medicine, Honda, Akita, Japan; and
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, USA
3Correspondence: Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: nsato{at}med.juntendo.ac.jp
SPECIFIC AIMS
Liver sinusoidal endothelial cells (SECs) possess unique receptors that recognize and internalize hyaluronic acid (HA). We used this characteristic and the unique assembling properties of comb-type polycations with DNA to develop a system for targeting foreign DNA to SECs.
PRINCIPAL FINDINGS
1. Synthesis of hyaluronate-grafted poly(L-lysine) copolymer
HA (Mn 3.2x105) was enzymatically digested by hyaluronidase to obtain low molecular weight fragments. The HA fragments (Mn 1.5x104) were conjugated to poly(L-lysine) (PLL) by a reductive amination reaction using sodium cyanoborohydride (NaBH3CN) as a reductant. The resulting comb-type copolymer (hyaluronate-grafted poly(L-lysine), PLL-g-HA) was isolated from unreacted HA fragments by dialyzing against 1 M NaCl through membranes with a molecular weight limit of 25,000 and lyophilized. Copolymer composition was determined from 1H-NMR spectra in D2O containing 500 mM NaCl.
2. Formation of the PLL-g-HA/DNA complex
PLL-g-HA was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Turbidity measurements confirmed that PLL-g-HA forms soluble nanoassociates with DNA. Agarose gel retardation assays revealed that the titration point representing the minimum proportion of PLL-g-HA required to retard DNA completely occurred at a 1:1 copolymer (based on PLL) to DNA (P/D) charge ratio, indicating selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains.
3. Stabilizing effects of PLL-g-HA on DNA duplex
In a UV-Tm profile of DNA duplex, the transition of 70°C at physiological ionic strength shows the melting of dsDNA to ssDNA. In the presence of excess DNA (poly(dA)·poly(dT)) over copolymer, only one transition was observed at 95°C. When the P/D charge ratio was 1.0, transition did not occur. Results indicate that the PLL-g-HA is effective in stabilization of duplex.
4. Uptake of PLL-g-HA/DNA by SECs
Under confocal microscopy, isolated SEC took up virtually no rhodamine isothiocyanate (RITC) -labeled DNA oligomers during incubation for up to 120 min. An increasing amount of DNA uptake was observed if DNA was included in culture media complexed with PLL-g-HA. Magnified images showed the presence of numerous bright red spots in cytosol of SECs, indicating localization of the internalized PLL-g-HA/DNA in the endosome/lysosome compartments. Fluorescein dextran added with the PLL-g-HA/DNA was localized to a different compartment of the SECs, suggesting the PLL-g-HA/DNA complex was internalized by SEC via a different mechanism from nonspecific endocytosis.
5. Targetability of the PLL-g-HA/DNA complex to liver SECs
Organ distribution of a 32P-radiolabeled plasmid (pSV ß-galactosidase expression vector [pSV ß-Gal]) injected via the tail vein of Wistar rats in equal quantities (7.5x106 counts) is shown in Fig. 1
a. When plasmid was complexed to PLL-g-HA, the PLL-g-HA/DNA complex was delivered almost exclusively to the liver. One hour after injection, >93% of the injected counts was detected in the liver. Residuals in other organs were 2.5% in intestine, 2% in spleen, 1.5% in kidney, and <<1% in heart, thymus, lung, and blood (Fig. 1a
). If DNA was complexed to PLL (Fig. 1a
) instead, the PLL/DNA complex was distributed mostly in the lungs. These results suggest the presence of HA is crucial for targeting the complex to the liver. Administration of the PLL-g-HA complexed to an FITC-labeled DNA revealed that the carrier–DNA complex was localized exclusively in the sinusoidal lining, which contains SECs (Fig. 1b
).
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6. Detection of gene expression in the liver transfected with the PLL-g-HA/pSVß-Gal
ß-Galactosidase mRNA could be detected by RT-PCR for up to 72 h after transfection in the group injected with the PLL-g-HA/pSV ß-Gal complex (Fig. 2
a, upper panel; lanes 2, 3, 5, 6, 8, 9). In contrast, no ß-galactosidase mRNA was detected even 24 h post-transfection when pSV ß-Gal was administered alone (Fig. 2a
, upper panel: lanes 4, 7, 10). In the absence of reverse transcription, pSV ß-Gal DNA was detected 24 h after transfection; no plasmid DNA was amplified beyond 24 h (Fig. 2a
, lower panel).
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Figure 2b
shows histological detection of cells expressing ß-galactosidase in the liver lobule 72 h after in vivo transfection. In the control in which pSV ß-Gal alone was administered, no cells expressing ß-galactosidase were seen (left panel). In contrast, a large number of SECs expressing ß-galactosidase was detected along the sinusoidal lining after transfected with an equal amount of pSV ß-Gal in a PLL-g-HA complexed form (right panel).
7. Stabilization of DNA triplex by PLL-g-HA
Effects of the PLL-g-HA on triplex DNA formation (purine and pyrimidine motifs) were analyzed by EMSA using a target duplex DNA (30 mer) from rat 
-1 collagen (I) promoter. In the presence of 150 mM KCl, complete inhibition of purine motif triplex formation was observed. The presence of the PLL-g-HA copolymer under the same conditions overcomes potassium inhibition.
pH dependence of pyrimidine motif triplex DNA severely limits its in vivo applications. Although stable pyrimidine motif triplex DNA was formed at pH 5.5, changing pH to 7.0 resulted in complete inhibition of its formation. The triplex-stabilizing efficiency of PLL-g-HA was significantly higher than that of oligocations like spermine and spermidine.
CONCLUSIONS AND SIGNIFICANCE
We have synthesized a new comb-type copolymer composed of HA and PLL. DNA migration into the agarose gel was completely retarded when DNA and the PLL-g-HA were incubated at a P/D charge ratio 
1, demonstrating that despite the presence of HA graft chains that bore negative charges, the PLL backbone can interact efficiently with DNA molecules. This selectivity of interpolyelectrolyte formation indicates that the negatively charged HA side chains of the PLL-g-HA copolymer remain free. This property is important, as the free HA chains are crucial for HA receptor-mediated delivery to the SEC (Fig. 3
).
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When distribution of PLL-g-HA complexed to 32P-labeled DNA was analyzed in different rat organs, the majority of radioactivity (>93%) was retained in liver (Fig. 1a
). Fluorescence derived from the PLL-g-HA/DNA complex detected linearly and homogeneously along sinusoidal lining cells (Fig. 1b
), indicating the selective delivery of the PLL-g-HA/DNA complex to the SEC. ß-Galactosidase expression was detected exclusively in SECs (Fig. 2b
).
Gene carrier systems composed of ligand molecules and polycations such as PLL have been reported. However, molecular conjugates composed of PLL and ligands are not stable or specific to target organs in vivo. When complexed to DNA, such conjugates are subject to precipitation because the PLL moiety forms insoluble complexes with DNA. Our data indicate that systemic application of a positively charged PLL/DNA complex resulted in >90% accumulation in the lung (Fig. 1a
).
A unique characteristic of the graft copolymer is that it contains hydrophilic polysaccharide side chains as a ligand (Fig. 3)
. PLL segments of the PLL-g-HA selectively interact with DNA, indicating that the PLL-g-HA/DNA complex forms a multiphase structure in which condensed PLL-DNA complex is surrounded by a hydrated hyaluronan-glycocalyx, a strategy that masks the PLL/DNA complex (i.e., "stealth" property) (Fig. 3)
. It is postulated that free HA chains on the complex prevent nonspecific uptake by major nonliver RES organs, leading to specific targeting of complexes to the liver (Fig. 1a
).
The ability to target specific sequences of DNA through oligonucleotide-based triplex formation provides a powerful tool for genetic manipulation. Triplex formation is, however, unstable in physiological conditions, which limits the strategy’s utility. It has been reported that comb-type polycations consisting of a PLL backbone and grafted chains of hydrophilic dextran stabilize triplex formation via regulation of the reversible transition of DNA by increasing the solubility of the complex and reducing the conformational changes of DNA. Our results indicate that changing the sugar moiety of the graft side chains to HA is as effective in stabilizing triplex DNA.
The PLL-g-HA system thus not only serves as a carrier of nuclear agents to the SEC, but stabilizes triplex DNA. We anticipate that the PLL-g-HA gene carrier system may lead to a new and more effective strategy to treat intractable liver diseases through manipulation of SEC functions by gene engineering.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0494fje; doi: 10.1096/fj.03-0494fje 
2 Both authors contributed equally to this work.
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