D Liu

University of Pittsburgh, Pittsburgh, PA, United States

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Publications (24)94.63 Total impact

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    ABSTRACT: A series of bifunctional compounds with galactosyl residues as targeting ligand for asialoglycoprotein receptors on hepatocytes and various dendrimers as the DNA-binding domain was synthesized. When mixed with plasmid DNA, these compounds self assembled into particles that exhibited high transfection activity both in vitro and in vivo. Optimal activity in liver cells was observed with compounds containing three galactosyl residues and 16 dendrimer arms. These results suggest that domain-based design is an effective strategy for development of a new generation of synthetic gene carriers.
    Gene Therapy 05/2007; 14(8):704-8. · 4.32 Impact Factor
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    ABSTRACT: The impact of hydrodynamic injection on liver structure was evaluated in mice using various microscopic techniques. Upon hydrodynamic injection of approximately 9% of body weight by volume, the liver rapidly expanded, reaching maximal size at the end of the injection and returned to its original size in 30 min. Histological analysis revealed a swollen appearance in the peri-central region of the liver where delivery of genes and fluorescence-labeled markers was observed. Scanning and transmission electron microscopy showed enlargement and rupture of endothelium that in about 24-48 h regains its morphology and normal function as a barrier against infection by adenovirus viral particles. At the cellular level in hydrodynamically treated animals, four types of hepatocytes were seen: cells with normal appearance; cells with enriched vesicles in the cytoplasm; cells with lightly stained cytosol; and cells with significant dilution of the cytoplasm. In addition, red blood cells and platelets were observed in the space of Disse and even inside hepatocytes. Vesicle formation is triggered by hydrodynamic injection and resembles the process of macropinocytosis. These results, whereas confirming the physical nature of hydrodynamic delivery, are important for a better understanding of this efficient method for intrahepatic gene and small interfering RNA delivery.
    Gene Therapy 02/2007; 14(2):129-37. · 4.32 Impact Factor
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    ABSTRACT: We have reported that a rapid tail vein injection of a large volume of plasmid DNA solution into a mouse results in high level of transgene expression in the liver. Gene transfer efficiency of this hydrodynamics-based procedure is determined by the combined effect of a large volume and high injection speed. Here, we show that the hydrodynamic injection induces a transient irregularity of heart function, a sharp increase in venous pressure, an enlargement of liver fenestrae, and enhancement of membrane permeability of the hepatocytes. At the cellular level, our results suggest that hepatic delivery by the hydrodynamic injection is accomplished by the generation of membrane pores in the hepatocytes.
    Gene Therapy 05/2004; 11(8):675-82. · 4.32 Impact Factor
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    T Ren, G Zhang, D Liu
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    ABSTRACT: Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.
    Bioorganic & Medicinal Chemistry 12/2001; 9(11):2969-78. · 2.90 Impact Factor
  • D Liu, J E Knapp
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    ABSTRACT: The need for a safe and efficient method for gene delivery has stimulated the recent emergence of vectorless methods (e.g., naked DNA) as promising alternatives to the available viral- and non-viral-based systems. Among these methods, hydrodynamics-based gene delivery through systemic injection of DNA offers a convenient, efficient and powerful means for high-level gene expression in mice. This review deals with the background and progress made so far with this new gene delivery method, the potential mechanism underlying the hydrodynamics-based DNA transfer into cells and its potential applications towards gene function analysis, protein expression and gene therapy in whole animals.
    Current opinion in molecular therapeutics 05/2001; 3(2):192-7. · 3.42 Impact Factor
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    G Zhang, Y K Song, D Liu
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    ABSTRACT: The liver is an important target organ for gene transfer due to its large capacity for synthesizing serum proteins and its involvement in numerous genetic and acquired diseases. Previously, we and others have shown that an efficient gene transfer to liver cells in vivo can be achieved by an intravenous injection of plasmid DNA using a hydrodynamics-based procedure. In this study, we systematically characterized the expression of transgene encoding a secretory protein in mouse. Using human alpha1-antitrypsin (hAAT) gene as a reporter, we demonstrate that the serum level of hAAT can reach as high as 0.5 mg/ml by a simple tail vein injection of 10-50 microg plasmid DNA into a mouse. The serum hAAT reaches the peak level 1 day after DNA injection and then declines during the following 2 to 4 weeks to 2-5 microg/ml, a level which persists for at least 6 months. Southern analysis of extracted DNA and RT-PCR analysis of RNA from the liver reveal that hAAT gene is active and present as episomal form after 6 months. These results suggest that the hydrodynamics-based transfection procedure provides a valuable tool for screening genes for therapeutic purposes in whole animals.
    Gene Therapy 09/2000; 7(15):1344-9. · 4.32 Impact Factor
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    T Ren, G Zhang, F Liu, D Liu
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    ABSTRACT: A new panel of steroidal cationic lipids has been synthesized for gene delivery. Using commercially available vitamin D2 (calciferol) or vitamin D3 (cholecalciferol) as hydrophobic motifs and a variety of cationic head groups as binding sites for negatively charged phosphate groups in DNA, we demonstrated that the transfection activity of the synthetic vitamin D-based cationic lipids 1d, 2d formulated with dioleoylphosphatidylethanolamine (DOPE) as a co-lipid is comparable to that of 3-(-[N-N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). These synthetic lipids are effective in transfecting a variety of cell lines. These results suggest that vitamin D-based cationic lipids are useful transfection reagents for in vitro gene transfer studies.
    Bioorganic & Medicinal Chemistry Letters 06/2000; 10(9):891-4. · 2.34 Impact Factor
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    ABSTRACT: Eleven structural analogues of two known cationic lipids, N-[1-(2, 3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) were synthesized and utilized to evaluate the structural characteristics of DOTMA for its high intravenous transfection activity. Using a CMV-driven expression system and luciferase gene as a reporter, the transfection activity of these analogues was evaluated in mice using tail vein injection. Results concerning the structure-activity relationship with regard to the influence of the backbone, relative position between head group and the hydrophobic chains on the backbone, linkage bonds, as well as the composition of the aliphatic chains revealed that cationic lipids which give a higher in vivo transfection activity share the following structural characteristics: (1) cationic head group and its neighboring aliphatic chain being in a 1,2-relationship on the backbone; (2) ether bond for bridging the aliphatic chains to the backbone; and (3) paired oleyl chains as the hydrophobic anchor. Cationic lipids without these structural features had lower in vivo transfection activity. These structural characteristics, however, did not significantly influence their in vitro transfection activity. The contribution that cationic lipids make to the overall in vivo transfection activity is likely to be determined by the structure of DNA/lipid complexes and by the outcome of the interaction between the DNA/lipid complexes and blood components upon intravenous administration.
    Gene Therapy 06/2000; 7(9):764-8. · 4.32 Impact Factor
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    ABSTRACT: A new series of cationic lipids has been synthesized for gene delivery using 3,5-dihydroxybenzyl alcohol as the backbone and starting material. Using CMV driven expression system and luciferase gene as a reporter, we demonstrated that the transfection activity of these new lipids when formulated with Tween 80 as co-lipid is comparable to that of DOTAP, one of the most commonly used cationic lipids for transfection. Among the four different cell lines tested including murine melanoma BL-6 cells, human embryonic kidney 293 cells, HepG2 and HeLa cells, the highest transgene expression was seen in 293 cells. Results from in vivo experiments using mice as an animal model show that these cationic lipids preferentially transfect the cells in the lung upon tail vein administration. The cationic lipid, N,N,N-trimethyl-N-[3,5-bis(tetradecyloxy)benzyl] ammonium bromide 4c(di-C14:0) with two 14-hydrocarbon chains exhibits the best transfection activity. These results suggest that these new aromatic ring-based cationic lipids are useful transfection reagents for both in vitro and in vivo gene transfer studies.
    Journal of Drug Targeting 01/2000; 7(4):285-92. · 2.77 Impact Factor
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    F Liu, Y Song, D Liu
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    ABSTRACT: Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.
    Gene Therapy 08/1999; 6(7):1258-66. · 4.32 Impact Factor
  • T Ren, D Liu
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    ABSTRACT: Quaternary ammonium lipids 1b-d, with diether linkages between hydrocarbon chains and butane or hexane backbone, were synthesized for cationic liposome-mediated gene delivery. The synthetic strategy of using C-4 or C-6 synthon permits the achievement of the variation of the hydrophobic domain as well as changes of space between the quaternary ammonium head and the hydrophobic domain in the diether-linked cationic lipids.
    Bioorganic & Medicinal Chemistry Letters 06/1999; 9(9):1247-50. · 2.34 Impact Factor
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    Y K Song, F Liu, D Liu
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    ABSTRACT: The effect of retention time of plasmid DNA in mouse lung on the level of transgene expression after intravenous administration was examined. Using CMV driven expression system with luciferase gene as a reporter and preinjection of free cationic liposomes into the animal as means of manipulating the retention time of plasmid DNA, we demonstrated that naked plasmid DNA is effective in transfecting cells in the lung by intravenous administration. An increase in DNA retention time in the lung results in a higher level of gene expression. Liposomes composed of cationic lipids with alkyl chains exhibited better activity than cholesterol-based cationic liposomes to retain the plasmid DNA in the lung. The level and patterns of gene expression obtained appeared similar to those seen in animals transfected by DNA-liposome complexes. These results suggest that prolonging the exposure time of DNA to the target cells in vivo may be an important strategy in achieving a high level of gene expression. Our data also introduce a possibility that the function of cationic liposomes in lipoplex-mediated transfection in vivo is to extend the interaction time of DNA with the cells.
    Gene Therapy 12/1998; 5(11):1531-7. · 4.32 Impact Factor
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    Y K Song, D Liu
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    ABSTRACT: Factors that regulate the transfection efficiency of cationic lipid-based carriers are still largely unknown. We have shown in a previous report [F. Liu, H.W. Qi, L. Huang, D. Liu, Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration, Gene Ther., 4 (1997) 517-523. ] that the transfection efficiency, to the lung, of a lipid formulation composed of N-[1-(2,3-dioleoyloxy)propyl-N,N, N-trimethylammonium chloride (DOTMA) and Tween 80 is directly proportional to the ratio of DOTMA to DNA. In this study, we investigated the mechanism underlying the high cationic lipid to DNA ratio dependent transfection activity. Specifically, we have examined the role of free cationic liposomes in affecting the transfection efficiency of the DNA/lipid complexes in vivo by intravenous administration. The data show that greater transfection activity of DNA/lipid complexes in the lung at a higher cationic lipid to DNA ratio is due to the function of free liposomes present in the DNA/lipid mixture. Free liposomes enhance the transfection activity of DNA/lipid complexes by increasing the retention time of DNA and decreasing transgene degradation in different organs. In addition to DOTMA liposomes, liposomes composed of 1,2-dioleoyl-3-trimethylammonium propane chloride (DOTAP) and 3beta[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) also enhance the level of gene expression in animals transfected by DNA/DOTMA complexes. These results suggest that inclusion of free liposomes into the DNA/lipid complexes may be important in achieving an optimal transfection activity in vivo.
    Biochimica et Biophysica Acta 07/1998; 1372(1):141-50. · 4.66 Impact Factor
  • Y K Song, F Liu, S Chu, D Liu
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    ABSTRACT: Physicochemical properties of the cationic liposomes, including structure of the cationic lipid-to-DNA ratio, liposome particle size, and inclusion of the helper lipids, were studied for their effect on the level, site, and duration time of gene expression in vivo by intravenous administration. Using a cytomegalovirus (CMV)-driven gene expression system containing either the luciferase or green fluorescence protein gene as a reporter and two cationic lipids [N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP)], we demonstrated in vivo by a single intravenous injection of DNA/liposome complexes into mice, that cationic liposomes are capable of transfecting cells in organs such as the lung, heart, liver, spleen, and kidney. Transfection efficiency is determined mainly by the structure of the cationic lipid and the ratio of cationic lipid to DNA. Although the presence of cholesterol in DOTAP liposomes did not affect transfection activity, inclusion of dioleoylphosphatidylethanolamine (DOPE) into either DOTAP or DOTMA liposomes significantly decreases liposome transfection activity in vivo. Results form time course show that gene expression in different organs is transient, with a peak level between 4 and 24 hr, dropping to less than 1% of the peak level by day 4. Experiments with repeated injections showed that the peak level of gene expression could be regained by subsequent injection.
    Human Gene Therapy 10/1997; 8(13):1585-94. · 4.02 Impact Factor
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    F Liu, H Qi, L Huang, D Liu
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    ABSTRACT: The factors controlling the transfection efficiency of cationic lipid carrier systems following intravenous administration are poorly understood. Using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) combined with Tween 80 as a carrier system and cDNA of luciferase or beta-galactosidase gene as a reporter, we investigated the importance of DOTMA to DNA ratio and the ratio of DOTMA to Tween 80 in the lipid formulation in determining the site and level of transgene expression following intravenous administration. The data show that all of the internal organs, including lung, liver, spleen, heart and kidneys, expressed the transgene upon systemic administration into animals with 25 micrograms of plasmid DNA when complexed with DOTMA-Tween 80 lipid formulation. The transfection efficiency was dependent on both DOTMA to DNA, and DOTMA to Tween 80 ratios. Among the organs examined, the lung appeared to be more transfectable than other organs. A better transfection activity was obtained with higher DOTMA to DNA and DOTMA to Tween 80 ratios. Time-response curve shows that gene expression was transient with a maximal level between 10 and 24 h after injection. Results from tissue distribution studies with 125I-labeled plasmid DNA and Southern analysis suggest that the transient expression is the result of the loss of transgene from the transfected cells. These results suggest that cationic lipid-based delivery systems can be efficient for gene delivery if the composition of the DNA-lipid complexes is properly controlled.
    Gene Therapy 07/1997; 4(6):517-23. · 4.32 Impact Factor
  • F Liu, J Yang, L Huang, D Liu
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    ABSTRACT: To develop appropriate dosage forms of DNA for gene delivery. 3 beta [N-(N', N' dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) was mixed either with Tween 80 alone, or with additional lipid components including castor oil and phosphatidylcholine (PC) or dioleoylphosphatidylethanolamine (DOPE) to make different lipid formulations. The particle size and the physical stability of the formulations upon mixing with plasmid DNA containing the luciferase cDNA were examined using laser light scattering measurement. The transfection activity of the DNA/lipid complexes was tested in presence or absence of serum using a cell culture system. We demonstrated that many favorable properties as a gene carrier could be achieved by formulating DNA into new dosage forms using Tween 80 as the major emulsifier. Compared to the cationic liposomes, these new formulations transfected different cell lines with an equivalent or higher efficiency. Not only are they resistant to serum, but also form stable DNA complexes which could be stored for longer periods of time without losing transfection activity. Cationic lipids formulated into different lipid formulations using Tween 80 as a surfactant appeared to have more favorable physical and biological activities than traditional cationic liposomes as a carrier for gene delivery.
    Pharmaceutical Research 01/1997; 13(12):1856-60. · 4.74 Impact Factor
  • F Liu, J Yang, L Huang, D Liu
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    ABSTRACT: To study the structure-function relationship of non-ionic surfactants in emulsion-mediated gene delivery. Four different types of non-ionic surfactants including Tween, Span, Brij and pluronic copolymers were used as co-emulsifiers for preparation of emulsions composed of Castor oil, dioleoylphosphatidylethanolamine (DOPE) and 3 beta [N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol). The effect of different surfactants on the formation of DNA/emulsion complexes and transfection activity were analyzed using plasmid DNA containing luciferase cDNA as a reporter gene. Non-ionic surfactants containing branched polyoxyethylene chains as the hydrophilic head group were more effective in preventing the formation of large DNA/emulsion complexes than those containing one or no polyoxyethylene chain. All emulsion formulations except those containing Brij 700 exhibited high activity in transfecting mouse BL-6 cells in the absence of serum. In the presence of serum, however, transfection activity of each formulation varied significantly. Emulsions containing Tween, Brij 72, pluronic F68 and F127 demonstrated increased activity in transfecting cells in the presence of 20% serum. In contrast to emulsions containing Span, long chain polyoxyethylene of Brij showed decreased transfection activity. The particle size of the DNA/emulsion complexes and their ability to transfect cells are dependent on the concentration of non-ionic surfactant in the formulation. The structure of the hydrophilic head group of the non-ionic surfactants in the emulsion is important in determining how DNA molecules interact with emulsions and the extent to which DNA is transferred inside the cell.
    Pharmaceutical Research 12/1996; 13(11):1642-6. · 4.74 Impact Factor
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    Q Hu, D Liu
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    ABSTRACT: The effect of serum on liver uptake of liposomes with different compositions was investigated using a single-pass liver perfusion technique. Among the liposomes tested are those containing CL, PA, DPGS, PE or glycolipids such as PI, GD, GT1b and aGM1. Liposomes containing PA, CL and DPGS showed high level of liver uptake in the absence of serum. Presence of serum decreased the total liver uptake for liposomes containing CL and PA by 50% and did not affect the level of liver uptake for DPGS-containing liposomes. The presence of serum, however, significantly increased the liposome uptake by the perfused liver for PG, PE and aGM1 liposomes. Liposomes containing PI showed a minimal liver uptake regardless of serum presence. Fluorescence microscopic studies using a dual fluorescence label system in combination with Kupffer cell elimination technique showed that, in addition to the dominant role of Kupffer cells in taking up liposomes, non-Kupffer cells may also be involved in taking up CL and DCP-containing liposomes. Competition experiments using various liposome compositions indicated that liposome uptake by the liver cells may involve different receptors. Serum activity in enhancing the liver uptake for PE- and aGM1-containing liposomes can be blocked by treatment of serum with EDTA, EGTA/Mg2+ and high temperature (56 degrees C), suggesting the involvement of complement system. Results from this study support the conclusion that blood clearance of liposomes by the liver involves two independent mechanisms, one requires serum opsonins and the other does not.
    Biochimica et Biophysica Acta 11/1996; 1284(2):153-61. · 4.66 Impact Factor
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    F Liu, D Liu
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    ABSTRACT: The rate of liposome clearance from blood by the reticuloendothelial system (RES), primarily the Kupffer cells of the liver, depends largely on liposome composition. Inclusion of phosphatidylserine or dicetyl phosphate into liposomes with a simple composition of phosphatidylcholine and cholesterol increases liposome clearance, while inclusion of GM1 or amphipathic poly(ethylene glycol) decreases the rate of liposome clearance. To understand the underlying mechanism by which liposome clearance is regulated by the RES, we have developed a simple liver perfusion system. Using mouse liver as a model, we demonstrated that hepatic uptake of neutral or negatively charged liposomes does not involve serum components. Liver uptake of liposomes is directly related to the surface characteristics of liposomes. Liposomes with a neutral composition of phosphatidylcholine and cholesterol exhibit relatively low liver uptake. Inclusion of PS or DCP into these liposome dramatically enhances liposome uptake by the perfused liver. Conversely, inclusion of GM1 or PEG derivatives into liposomes greatly reduces the liposome uptake by the mouse liver. In contrast to the neutral or negatively charged liposomes, serum enhances the liver uptake of positively charged liposomes. Such serum effect on liver uptake of the positively charged liposomes is likely due to liposome aggregations caused by serum proteins. Inhibition of the liver uptake for PS-containing liposomes using liposomes with different compositions suggests that liver uptake of liposomes may involve different receptors.
    Biochimica et Biophysica Acta 02/1996; 1278(1):5-11. · 4.66 Impact Factor
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    D Liu, Q Hu, Y K Song
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    ABSTRACT: The kinetics of blood clearance and the mechanisms of liposome uptake by the reticuloendothelial system (RES) were compared in two animal species (mice and rats). By employing an in situ liver perfusion technique with selected liposome compositions (PC/Chol, PC/Cho/PS, PC/Chol/GM1 and PC/Chol/PEG5000-PE), we demonstrated that liposomes with same lipid composition exhibited different blood circulation half-lives in different animal species. Although liver is the major organ responsible for the clearance of liposomes from blood in both animal species, the specific mechanisms differ. In mice, liposome uptake by the liver did not involve specific serum opsonins. In contrast, liposome uptake by the rat liver was strongly dependent on serum opsonins. Further, the activity of serum opsonins for a given liposome composition differed among animal species. Human serum exhibited higher opsonin activities for PC/Chol and PC/Chol/GM1 liposomes than bovine sera, while rat serum displayed a high opsonizing activity for GM1 liposomes and none for liposomes composed of PC and Chol. The opsonin activity of human serum could be removed or decreased by treatment with EGTA/Mg2+, EDTA or cobra venom factor, suggesting that the activity is likely due to complement components. It is likely that C3 of the human complement system plays an important role in mediating the uptake of liposomes by the liver.
    Biochimica et Biophysica Acta 01/1996; 1240(2):277-84. · 4.66 Impact Factor