Hepatocytes targeting of cationic liposomes modified with soybean sterylglucoside and polyethylene g

PO Box 2345, Beiing 10023, ChinaWorld I Gastroentero! 2005;11(32):4947-4952www.wjgnet.comWorld. Journal of Gastroenterology ISSN 1007-9327wig@wignet. comELSEVIER◎2005 The wjG Press and Elsevier Inc. All rights reserved.●BASIC RESEARCH●Hepatocytes targeting of cationic liposomes modified with soybeansterylglucoside and polyethylene glycolXian-Rong Qi, Wen-Wei Yan, Jing Shi .Xian-Rong Qi, Wen-Wei Yan, Jing Shi, Departnent of Pharmaceutics,INTRODUCTIONSchool of Pharmaceutical Sciences, Peking University, BejjingCationic liposomes have been accepted as effective non-viral100083, ChinaSupported by the National Natural Science Foundation of China,vectors for genc dclivery with a lower immunogenicity thanNo. 30371265the viral ones. However, the lack of organ or cell specificityCorrespondence to: Professor Xian-Rong Qi, Department ofsometimes hampers their applications. In the case of cationicPharmaceutics, School of Pharmaceutical Sciences, Peking University,liposomes, the highest gene expression is observed in theBeijing 100083, China. qixr2001 @ yahoo.com.cnlung after intravenous injection of their plasmid DNATelephone: +86-10-82801584 Fax: +86- 10- 62015584complexes in most cases, because the lung capillaries are theReceived: 2004-11-15 Accepted: 2005 02-18first traps to be encounteredlA. Development of cell- specifictargeting technology for cationic liposomes attracts greatinterest in gene therapy.AbstractSince the liver is one of most important target organs inthe body, and Kupffer cells in the liver are a part of the reticularAIM: In this study, a hepatocyte-specific targeting technologyendothelial system (RES), relatively high accumulation ofwas developed by modifying cationic liposomes with soybeanadministered liposomes is observed in the liver, mostly insterylglucoside (SG) and polyethylene glycol (PEG) (C/SG/non-parenchymal ells'l. However, preferential incorporationPEG-liposomes).of liposomes into liver hepatocytes is required for therapeuticMETHODS: The liposomal transfection efciencies in HepG2situations. Thus, reducing the Kupffer cell uptake and2.2.15 cells were estimated with the use of fluoresceinenhancing hepatocyte uptake, are challenges of research insodium (FS) as a model drug, by flow cytometry. Theliposome -targeting.antisense activity of C/SG/PEG-liposomes entrappedFor cell specific delivery, the receptor mediated endocytosisantisense oligonucleotides (ODN) was determined assystems endowed to various cell types would be useful andHBsAg and HBeAg in HepG2 2.2.15 cells by ELISA. Thea number of gene delivery systems have been developed toliposome uptake by liver and liver cells in mice was carriedintroduce gene into specific cells with receptor -mediatedout after intravenous injection of 3H-labeled liposomes.endocytosis. Ligands currently being investigated includegalactosel, lactoselo, transferring", etc. Among these recRESULTS: C/SG-liposomes entrapped FS were effectivelyasialoglycoprotein receptor (ASGP-R) is the most promisingtransfected into HepGz 2.2.15 cels in vitro. C/SG/PEG-for gene targeting, since it exhibits high affinity and a rapidliposomes entrapped ODN, reduced the secretion ofinternalization rate8.both HBsAg and HBeAg in HepG2 2.2.15 cells whenIt was reported that liposomes with soybean sterylgl-compared to free ODN. After in vivo injection of 3H-labeleducoside (SG) gets accumulated in the liver, especially inC/SG/PEG-liposomes, higher radiation accumulation washepatocyteslP.10. Doxorubicin (DXR) entrapped in liposomesobserved in the hepatocytes than non-parenchymal cellscontained SG (SG-liposomes) showed a high therapeuticof the liver.effect for its selective delivery of drugs to hepatocytes inCONCLUSION: C/SG/PEG-liposomes mediated geneanimals with liver cancerll. SG-liposomes have glucosetransfer to the liver is an effective gene-delivery methodresidue on the surface of liposomcs12), which is essentialfor hepatocytes- specific targeting, which appears to havefor selective accumulation in liver cells.Hepatitis B is a disease of global importance with morea potential for gene therapy of HBV infections.than 300 million carriers of the hepatitis B virus (HBV)@ 2005 The WJG Press and Elsevier Inc. All rights reserved.worldwidel1J. Unfortunately, treatment of chronic HBVinfection is far from satisfactory. The most successfulKey words: Hepatocytes targeting; Cationic liposomes;therapeutic agent so far available is interferon alpha, whichSoybean sterylglucosideshowsints after completion ofthe the中国煤化工have becoe scsQi XR, Yan WW, Shi J. Hepatocytes targeting of cationictargets|YHC N M H Gategy may be promisingliposomes modified with soybean sterylglucoside andin targeung cnronic H1D v Inrecoon, and several studies havepolyethylene glycol. World J Gastroentero/ 2005; 11now shown that ODN are capable of suppressing HBV(32): 4947-4952in virol6.17] and in viovl8.9. ODN are synthetic single chainhttp://www.wjgnet.com/1007-9327/11/4947 .aspDNA molecules that can inhibit gene expression within cells.4948ISSN 1007-9327 CN 14-1219/ R World J GastroenterolAugust 28, 2005 Volume 11 Number 32by their capability to bind a complementary mRNA sequence,study were prepared according to the compositions in Table 1,and prevent translation of mRNA, thus providing potentiallyrespectively. A mixture of lipids in chloroform was driedpowerful therapeutic tools against viral diseases and cancerP20under a stream of nitrogen and additionally dried underFor an ODN delivety system towards HBV infection, thevacuum for 3 h to remove all chloroform. The dry lipidSG may be useful for targeting hepatocytes of the liver.film was resuspended with FS or ODN solution (solutionThis study describes a specific targeting approach whichin 50 g/L glucose) by vortexing and sonicating, and thenresults in increased hepatocytes uptake. A cationic liposomeextruded through 0.2 μm pore size polycarbonate filters tocarrier modified with SG and encapsulated 15-mer ODNgencrate the FS and ODN encapsulatcd liposomes,for the HBV therapy in viro was constructed. The influencerespectively. The concentration of lipids was 20 umol/L.of SG on facilitating the uptake of liposomes by hepatocytesTo prepare lipid-radioactive labeled liposomes, H-Ch waswas investigated in vivo. The value of the surface modifiedadded to the lipid mixture at the beginning of the liposomescationic liposomes as a delivery vehicle, mainly forpreparation and the dry lipid film was resuspended withhepatocytes targeting antisense agent in vitro and in vrivo was5% glucose. The H labeled C-liposomes and C/SG/PEG-assessed.liposomes were prepared. The radioactivity of liposomeswas 4 μC/200 μLMATERIALS AND METHODSDetermination of encapsulation fficacy of liposomesSynthesis of ODNFree ODN was separated from ODN encapsulatedODN with phosphorothioate backbone encoding the capliposomes by equilibrium dialysis, in a dialysis tubingsite of SP II promoter transcribed mRNA (cap site/SP II)(SpcraPor 12 000 to 14 000 MWCO)at4 C for 12h insequences were synthesized using standard phosphoramadite10 mL of 5% glucose solution. The incubation liquid waschemistry by Aoke (Beijing, China) and purified by SDS-taken and the concentration of ODN was detected by UVPAGE. The complementary ODN sequences were: 5' GATspectro photomcter at 260 nm. Free FS was scparated fromGAC TGT CTC TTA 3'.encapsulated FS by passing through a Sephadex G-50column (1 cmx20 cm). The concentration of FS wasAnimals and cell linedetermined by measuring the fluorescence intensity of FSMale KM mice (18-23 g) were obtained from the Institutewith excitation and emission wavelengths at 490 andof Zoology, Chinese Academy of Sciences Beijing, China).512 nm, respectively. According to the amount of ODNAll animals received good care. A human hepatoblastomaor FS entrapped in the liposomes, the encapsulationcell line, HepG: 2.2.15 was provided by the Institute of efficiency was calculated.Hepatology of the People's Hospital, Peking University(Beiing, China).Morphology and size analysisThe size of liposomes was determined by dynamic lightMaterialsscattering using a Zetasier 3 000HS (Malvern Instruments,N, N-dimethylethylenediamine (99%) and cholesterylLtd, UK). The morphologies of these liposomes were alsochloroformate (97%) were obtained from ACROS (USA); .observed by the transmission electron microscope.dipalmitoylphosphatidylcholine (DPPC) and polyethyleneglycol- distcaroylphosphatidylethanolamine (PEG-DSPE)Cell culture and transfection efficiency measurementwere purchased from NOF (Tokyo, Japan); sG was generouslyHepG; 2.2.15 was maintained in DMEM mediumsupplicd by Ryukakusan Co. Ltd. (Tokyo, Japan); cholesterolsupplemented with 100 mL/L FBS at 37 °C with 50 mL/L(Ch) was purchased from Wako Pure Chemical IndustriesCO2. The cells were scraped by 0.25% trypsin and planted(Tokyo, Japan); H-Ch and 125I-ODN was provided by Chinain 96-well tissue culture plates (5x 103/well) for 2 d beforeInstitute of Atomic Energy (Beiing, China). FS was obtainedthe experiment, until the percentage of adherent cellsfrom the Third Chemical Reagent Factory of Shanghaircached approximately 70% confluence. The upper medium(Shanghai, China); DMEM medium and fetal bovine scrumwas removed and fresh DMEM medium was added with(FBS) was purchased from Life Technologies (NY, USA).100 mL/L FS encapsulated liposome. When the liposomes2, 5- Diphenyloxazole (PPO) and 1, 4bis (5 pheny)-2-oxazolyD-were incubated with cells for 3, 6, and 24 h, respectively,benzene (POPOP) were provided by Fluka (Buchs, Switzedland).the cells werc detached with 0.25% trypsin and washedCollagenase (I) was putchased from Sigma (St. Louis, MO,thrice with 10 mmol/L PBS. The transfection efficiencyUSA). All other chemicals were of reagent grade.was determined by counting the amount of cells transferredby FS with flow cytometry (BD, USA). The transfectionSynthesis of DC-cholefficiency was calculated according to the followingDC-chol was synthesized according to the methodequation: amount of FS transferred cells/ amount of totaldescribed by Gaol21l. The production was confirmed bycells>中国煤化工ection efficiency werethin-layer chromatogram (ILC), melting point, 'H nuclearcalcul{YHCNMHGxperiments.magnetic resonance (NMR) (500 MHz, CDCI3), massspectrum (MS), etc.Antisense activity of ODN encapsulated in liposomesFor lipofection, HepG2 2.2.15 cells werc sceded at an initialPreparation of liposomesconcentration of 1x10* per well for 96 well plates. TheThe FS or ODN encapsulated liposomes used in the presentcells were alowed to grow for about 24 h, until the percentageQi XR et al. Hepatocytes targeting of SG/PEG-liposomes4949of adherent cells reached approximately 80% confluence.entrapped in the N-liposomes, with an entrapment efficiencyThen the cells were washed extensively to remove theonly at 0.64%. By adding DC- chol to the N-liposomes, thepreviously secreted HBsAg and HBcAg in the medium. Afterentrapment efficiencies of FS and ODN were increased towashing, 100 uL free ODN or C/SG/PEG-liposomesmore than 83%, when the charge ratio of anionic FS orentrapped ODN with an ODN concentration at 1.25, 2.5 .ODN/ cationic lipid was 1:1. The C-liposomes, C/SG-or 5.0 μmol/L, together with 100 μL DMEM containingliposomcs and C/SG/PEG liposomes had small size (155.0,10% serum, were added. The secretion of HBsAg and117.8, and 96.2 nm for FS entrapped in liposomes, 71.0,HBeAg into the culture supernatants was measured daily75.4, and 183.0 nm for ODN entrapped in liposomes,for 3 d, using ELISA immunoassay kits. The means ofrespectively). The polydispersity index indicated theHBsAg and HBeAg immunoassay measurements wereuniformity in size distribution. Transmission electroncalculated from two independent experiments.micrographs of these liposomes showed spherical vesicles(data not shown).Liver uptake in vivo and scinillation countingLiposomes labeled with 3H-Ch (C- liposomes and C/SG/Transfection eficiency of liposome in HepG222.15 cellsPEG-liposomes) were injected into the tail vein of threeFigure 1 showed the transfection of FS cntrapped in N-male mice with a dose of 200 μL/20 g. At 0.5 and 4 h afterliposomes, C-liposomes and C/SG-liposomes when theyinjection, the mice were killed. The liver tissue was collectedwere incubated for 3, 6, and 24 h with HepGz 2.2.15 cells .and washed with saline. About 100 mg of liver samplesat 37 C, respectively. FS served as a fluorescent markerwere decolored in a solution, containing 200 μL HClO,because the cell uptake of free FS was almost negligible.and 300 μ 60% H2O2. Radiation scintillation fluid wasAs shown in Figure 1A, the transfection of C-liposomesadded and mixed thoroughly. The radioactivity (dpm) ofentrapped FS was significantly higher than that of N-samples was counted on a scintillation counter Pharmacialiposomes, and the transfection of C/ SG-liposomes entrappedWALAC 1410, Turku, Finland).FS was the highest. The enhancement of transfectionefficiency was about 7.7-fold for the C/SG liposomes thanlsolation of liver cllsthat of C-liposomes when incubated with cells for 6 h. WhenMice were injected intravenously by tail vein with"H-labeleddifferent types of liposomes were incubated with HepGzC-liposomes and C/SG/PEG-liposomes, respectively. At2.2.15 cellsat37 °C for 24 h, the C/SG-liposomes showed0.5 and 4 h after administration, the mice were anesthetized,the highest transfection efficiencies, while the C liposomesand the liver was perfused via the portal vein with isotonicand C/ PEG-liposomes did not show significant differencessaline to remove the blood. Then the liver was excised,(Figure 1B).minced and digested in 0.5 g/L collagenase for 30 min at37 C. The suspended cells were filtered through cottonEffect of cationic liposomes entrapped ODN on HepGz 2.2.15mesh sieves, followed by centrifugation at 500 τ/ min forexpression3 min. The pellets containing hepatocytes were washed thriceFigure 2 showed the effect of cellular treatment with freewith saline solution by centrifugation at 500 r/min forODN and C/SG/PEG-liposomes entrapped ODN on the3 min. The supernatant containing non-parenchymal cellswerc similarly centrifuged and washed thrice at 1 500 r/ minexpression level of HBsAg and HBeAg protein in HepG2 .for 15 min. The radioactivity (dpm) of hepatocytes and2.2.15 cells. The antisense effect (percent of inhibition onnon-parenchymal samples was counted on a scintillationHBsAg and HBeAg secretion) of ftee ODN and C/SG/PEG-liposomes entrapped ODN was affected by thecounter, as described previously.concentration of ODN and the cultured times. When theconcentration of ODN that was added increased fromRESULTS .1.25 to 5.0 umol/L, the inhibition percentage of HBsAgCharacteristics of liposomesby C/SG/PEG-liposomes entrapped ODN increased fromThe entrapment efficiencies, size, polydispersity index of59.15% to 90.37% at 72 h, meanwhile, the inhibition ofall kinds of FS or ODN encapsulated liposomes are shownHBeAg increased from 42.9% to 73.43%. The inhibitionin Table 1. The results indicated that FS could hardly beeffects of free ODN on HBsAg and HBeAg were decreasedTable 1 Entrapment efficiencies (EE), mean intensity diameter (D) of vesicle and polydispersity index (PI) of FS entrapped in liposomes andODN entrapped in liposomesLiposome compositionsEntrapment of FSEntrapment ofODNSample(molar ratio)EE(%) .D(nm)PINiposomesDPPC/Ch(10:10)0.64中国煤化工CliposomesDPPC/Ch/DC-chol (10:1:10)88.58+4.48'155.0MYHCNMHG1.00C/SG-liposomesDPPC/C/DC-dhol/SG(10:1:10:134)88.46+2.2910.310.44C/SG/PEC-liposomesDPPC/Ch/DC- chol/SG/PEG-DSPE (10:1:10:134:134)83.12+3.63296.20.2289.54+1.24-183.00.35'Values represent as mean tSD,n= 4. 2Values represent as mentSD,n= 3..4950ISSN 1007-9327 CN 14-1219/ R World J GastroenterolAugust 28, 2005 Volume 11 Number 32A50「B 235r25 t205|-片13 121620 24Time (h)Figure1 Transfection eficiencies. A: Transfectionefiecies ofF; entrappppedC/SG-iposomes. Data represents the average of two wells; B:in dfferent types of liposomes incubated with HepG2 2.2.15 cellsat37 C...0-: free FS solution as control; -●-: N-liposomes; .▲: C-liposomes;with HepG22215 Cells at 37 C for 24 h. Data represents mean+SD. n= 3.n=3.100100 rC0t30 F30 -0F50 t10 FH0 F4020 t20 F0g 2040 60 800204060800%20406080Figure 2 Percent of inibitioi on HBsAg and HBeAg secretin of free ODN and_: free ODN; --. C/SG/PEGliposomes encapsulated ODN; 0: HBsAg;C/SG/PEG-liposomes entrapped ODN in HepGz 2.2.15 cells culture medium▲: HBeAg. Experimental values are given as the mean of two independentincoubated at 37 C. The ODN concentration was 1.25 (A), 2.5 (B) and 5.0 umoL(C),experiments.and the inhibition efcts ofC/SG/PEG-liposomes entrapped(total hepatocyte and non-parenchymal cells) was notODN were increased when incubation time was increasedsignificantly different for C-liposomes and C/ PEG/SG-from 24 to 72 h. The inhibition on HBeAg secretion broughtliposomes. The uptake amounts of liposomes at 0.5 and 4 hby frce ODN and C/SG/PEG liposomes entrapped ODNin hepatocytes and non-parenchymal cells are also shownshowed lowcr tendency compared to the HBsAg. The cellsin Figure 3. After separating liver cells into hepatocytes andremained viable throughout the experiments and nonon-parenchymal cells, it was found that the uptake ofmorphological abnormalities were observed.C/SG/PEG-liposomes was higher than that of C-liposomesby hepatocytes at 0.5 h (P<0.01) and the uptake of C/SG/ .Distribution in liver and intrahepatic cells in vivoPEG liposomes was lower than that of C-liposomes by non-H Ch labeled C-liposomes and C/SG/PEG -liposomes wereparenchymal cells at 0.5 and 4 h (P<0.01). These resultsinjected in mice at a dose of4 μCi/20 g. At 0.5 and 4 h .indicated that the C/SG/PEG-liposomes have moreafter injection, the radioactivity in 100 mg of liver tissue isappetency to hepatocytes than non-parenchymal cells inshown in Figure 3. The distribution amount in liver tissuethe liver.A112 000B6000r10 0005 000导800，4 0006 0003 000尊4 000中国煤化工b2 000MHCNMHG0LLiver tissueHepatocyte Non-parenchymalHepatocyteNon-parenchymalFigure 3 Distribution in liver tissue and uptake amounts in hepatocytes andrepresents C-liposomes and the flled bar represents C/PEG/SG-liposomes.non-parenchymal cells of liver of Habelled lposomes, after intravenous administrationData represent mean+SD, n= 3. The statistical signilicance of differences wasby til vein-te iifterent time periods. A:0.5; B: 4.0h. The open bar analyzed by Student's test. P<0.01 Vs CIPEG/-liposomes.Qi XR et al. Hepatocytes targeting of SG/PEG- liposomes4951DISCUSSIONeffciency of C/SG/PEG liposomes entrapped ODN alsoincreased (Figure 2). However, it is demonstrated that largerliposomes seem to be promising, because of their high geneamounts of ODN and cationic lipid would lead to a higherexpression efficiency. When using simple cationic liposomestoxicity to the cells; therefore, concentration of liposome-by both intravenous and intraportal administration19.2, it is .ODN complexes should be restricted at a certain level24.difficult to transfect into hepatocytes because liposomesAs for the present ODN encapsulated C/SG/PEG-liposomes,prefer targeting the lung and RES. A great challenge facesa concentration of DC-chol lower than 10 ug/ mL with athe investigator who wishes to target liposomes to hepatocytes,cationic lipid/ODN at 1:1 of charge ratio was regardedfor some disorders such as hepatitis B or metabolic diseasessuitable.which require that the liposomes be stcered away from theirIn the study of liver uptake of 'H labeled cationicnatural targets. In this study, the characteristics of SGliposomes, no significant difference was found between C-modified liposomes and its inhibition on HBsAg and HBeAgliposomes and C/SG/PEG-liposomes. These resultssecretion was investigated. In addition, the liver uptake andindicated that the PEG chain and glucose residue in SG didintrahepatic distribution of a labeled cationic liposome andnot necessarily improve the in vivo behavior of cationicSG modified cationic liposome were also evaluated.liposomes. Kirby et al, showed that when the cationicTo investigate the effect of DC-chol, SG and PEG DSPEliposomes contain only 5% of charged lipid with small zetaon the entrapment and transfection efficiency, FS was usedpotential, the behavior of the cationic liposomes are notas a marker. DC- chol (positively charged) can bind with FSdifferent from that of neutral liposoms12.25. Our resultsor ODN (both negatively charged) by electrostatic interaction.agree with these findings. These results could be attributedTypical cationic liposomes that carry excess positive chargeto the special hepatocytes targeting behavior of SG. Awill interact with plasma proteins, and would be rapidly takenprevious study showed that the glucose residue on the surfaceup into the mononuclear phagocytic system. In order toof liposomes could selectively recognize the ASGP-R ofdecrease the adsorption of plasma proteins and interactionhepatocytcs cells?!. The result also demonstrated that thewith non-target cells, 6% PEG-DSPE was added into theliver targeting effect of SG is not diminished by the PEGcationic liposomes.chain on the surface of liposomes.Entrapment of FS and ODN to cationic liposomes wasIn conclusion, this study established a highly efficientvery efficient, even when containing a relatively high amountreceptor- mediated delivery system for ODN to hepatocytesof SG and PEG-DSPE (6%, Table 1). Evidently, SG andby using SG and PEG modified cationic liposomes. By utilizingPEG coating seldom shields the positively charged liposomethis delivery system, an ODN targeting the encapsidationsurface ftom interaction with FS and ODN.site of the HBV pregenome causes a strong inhibition ofReceptors for carbohydrates such as the ASGP-R onHBV replication in viro. Therefore, SG modified liposomeshepatocytes and the mannose receptor on macrophages andmay be effective vehicles to improve the delivery of ODNliver endothelial cells produce opportunities for cell-specificto the liver for the therapy of hepatotropic viruses.gene delivery with liposomal carriers. The presence of aglucoside on the surface of etrically ncutral FS entrappedACKNOWLEDGMENTS .in C/SG-liposomcs, resulted in more than a 2- fold increasedWe would like to thank Professor Lai Wei and his researchtransfection efficiency of HepGz cells, which is a humangroup at the Institute of Hepatology, Pecking University forhepatoblastoma cell line that is known to express ASGP-R,assistance in cell culture and transfection efficiencywhen compared to C-liposomes encapsulated FS (Figure 1).measurement.It was surmised that such glucoside in SG could be identifiedby ASGP-R present on the surface of the HepGz clls, leadingto liposome entry into cells through endocytosis.REFERENCESThe major HBsAg, and in some cases, the HBeAg, isZhu N, Liggitt D, Liu Y, Debs R. Systemic gene expressiondetectable in the serum of individuals with chronically infectedafter intravenous DNA delivery into adult mice. Science 1993;9211HBV. Serological detection of HBeAg usually correlates261: 209-211well with the presence of circulating virions and is commonly2 Huang L, Li s. Liposomal gene delivery: a complex package.Nat Biotechnol 1997; 15: 620-621used clinically as an indicator of active HBV replication.3 Dasgupta P, Bachhawa BK. Receptor mediated uptake ofElimination of HBsAg and HBeAg from the supernatant isasialognglioside liposomes: sub-cellular distribution of theassociated with resolution of infection with a wild-type strainliposomal marker in isolated liver cell types. Biochem Int 1985;of HBV. Synthetic ODN (15-mers), complementary to the10: 327-336cap site of SP II promoter transcribed mRNA, showed a4 Hara T, Aramaki Y, Takada S, Koike K, Tsuchiya s. Receptor-mediated transfer of pSV2CAT DNA to a human hepatoblastomasequencc-specific, dose-dependent inhibitory effect on HBVcell line HepG2 using asialofetuin-labeled cationic liposomes.gene expression from a concentration of 1.25- 5.0 umol/L.A previous study showed that, such ODN could inhibit the中国煤化工Schuber F Behr JP. Tar.expression of HBsAg without a significant effect on totalcells with lipopolyamine-synthesized protein in the cells[2I. According to the resultsYHC N M H G galactose ligands: a stagetoward artiticial viruses. Proc Natl Acad Sci USA 1995; 92:of this study, it is found that the initial concentration of1744- 1748ODN and incubation time appeared to be important factors6 Choi YH, Liu F, Park JS, Kim Sw. Lactose-poly-(ethylenein obtaining a desired inhibition effect. As the ODNglycol)-grafted poly-L-lysine as hepatoma cell-targeted geneconcentration and incubation time increased, the inhibitioncarrier. Bioconjug Chem 1998; 9: 708-7184952ISSN 1007-9327 CN 14-1219/ R World J GastroenterolAugust 28, 2005 Volume 11 Number 327 Kircheis R, Kichler A, Wallner G, Kursa M, Ogris M, Felzmann3021-3025T, Buchberger M, Wagner E. Coupling of cell-binding ligands7 Wu GY, Wu CH. Specific inhibition of hepatitis B viral geneto polyethyleneimine for targeted gene delivery. Gene Therexpression in vitro by targeted antisense oligonucleotides. I1997; 4: 409-418Biol Chem 1992; 267: 12436-124398 Wagner E. Application of membrane-active peptides for18 Offensperger WB, Offensperger S, Walter E, Teubner K, Igloinonviral gene delivery. Ado Drug Del Rev 1999; 38: 279- -289G, Blum HE Gerok W. In vivo inhibition of dack hepatitis BShimizu K, Maitani Y, Takayama K, Nagai T. Evaluation ofvirus replication and gene expression by phosphorothioatedipalmitoylphosphatidylcholine liposomes containing a soy-modified antisense oligodeoxynucleotides. EMBOI 1993; 12:bean-derived sterylglucoside mixture for liver targeting. ) Drug1257-1262Target 1996; 4: 245-25319 Soni PN, Brown D, Saffle R, Savage K, Moore D, Gregoriadis G,10 Shimizu K, Maitani Y, Takayama K, Nagai T. FormulationDusheiko GM. Biodistribution, stability, and antiviral efficacyof liposomes with a soybean-derived sterylglucoside mixtureof liposome-entrapped phosphorothioate antisenseand cholesterol for liver targeting. Biol Pharm Bull 1997; 20:oligodeoxynucleotides in ducks for the treatment of chronic duckhepatitis B virus infection. Hepatology 1998; 28: 1402-141011 Shimizu K, Qi XR, Maitani Y, Yoshi M, Kawano K, Takayama20 Wagner RW. Gene inhibition using antisense oligodeoxy-K, Nagai T. Targeting of soybean-derived sterylglucosidenucleotides. Nature 1994; 372: 333-335liposomes to liver tumors in rat and mouse models. Biol Pharm21 Gao X, Huang L. A novel cationic liposome reagent for effi-Bull 1998: 21; 741-746cient transfection of mammalian cells. Biochem Biophys Res2 Shimizu K, Maitani Y, Takayama K, Nagai T. Characteriza-Commun 1991; 179: 280-285tion of dipalmitoylphosphatidylcholine liposomes contain-22 Ross PC, Hui Sw. Lipoplex size is a major determinant of ining a soybean-derived sterylglucoside mixture by differentialvitro lipofection efficiency. Gene Ther 1999; 6: 651-659scanning calorimetry, Fourier transfosform infrared spectroscopy,23 Li S, Huang L. In vivo gene transfer via intravenous adminis-and enzymatic assay. J Pharm Sci 1996; 85: 741-744tration of cationic lipid-protamine-DNA (LPD) complexes.13 Maynard JE. Hepatitis B: global importance and need forGene Ther 1997; 4: 891-90014 Hoofnagle JH. Chronic hepatitis B. New Engl J Med 1990;liposomes by cultured mouse bone marrow macrophages: .323: 337-339influence of liposome composition and size. Biochim Biopiys5 Dusheiko GM. Treatment and prevention of chronic viralActa 1991; 1061: 56-64hepatitis. Pharmacol Ther 1995; 65: 47-7325 Huang sK, Martin F, Friend DS, Papahadjopoulos D. Mecha-16 Goodarzi G, Gross SG, Tewari A, Watabe K. Antisensenism of stealth liposome accumulation in some pathologicaloligodeoxyribonucleotides inhibit the expression of the gene fortissues, In: D. Lasic, F. Martin (Eds.), Stealth Liposomes,hepatitis B virus surface antigen. J Gen Virol 1990; 71(Pt 12):Boca Raton CRC Press FL 1995: 119-125Science Editor Wang XL and Guo sY Language Editor Elsevier HK中国煤化工MYHCNMHG