mechanisms of co-modified liver-targeting liposomes as gene delivery carriers based on cellular...

117 (2007) 281–290www.elsevier.com/locate/jconrel N

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Journal of Controlled Release

GE

Mechanisms of co-modified liver-targeting liposomes as gene deliverycarriers based on cellular uptake and antigens inhibition effect

Yuan Zhang a, Xian Rong Qi a,⁎, Yan Gao b, Lai Wei b, Yoshie Maitani c, Tsuneji Nagai c

a School of Pharmaceutical Sciences, Peking University, Beijing 100083, Chinab Institute of Hepatology, Peking University People's Hospital, Beijing 100044, China

c Institute of Medicinal Chemistry, Hoshi University, Ebara 2-4-41, Shinagawa-Ku, Tokyo 142-8501, Japan

Received 26 March 2006; accepted 9 November 2006Available online 16 November 2006

Abstract

In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, theliver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor(ASGPR), β-sitosterol-β-D-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linkedimmunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strongantigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different stepsduring the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated throughantigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiencyseemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis,the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfectionbut no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effectivecarrier to transfer asODN into hepatocytes infected with HBV orientedly.© 2006 Elsevier B.V. All rights reserved.

Keywords: β-sitosterol-β-D-glucoside; Liver-targeting; Co-modified cationic liposomes; Membrane fusion; Endocytosis

1. Introduction

In order to transfer the therapeutic asODN effectively into thecells both in vitro and in vivo, various gene carriers have beendeveloped by many groups [1,2]. Among those, the cationicliposomes is one kind of widely used non-viral carriers with hightransfection efficiency in vitro, convenient availability, and goodflexibility which allows modifications to meet different require-ments. However, for successful in vivo applications, it still needsto be improved in the aspects of transfection efficiency,cytotoxicity, targeting delivery, half-life time, etc. [3,4].

Many studies have confirmed the anti-HBV effects ofdifferent asODN, and as a new prospective method, anti-HBVgene therapy has attracted much attention [5–7]. Since HBV

⁎ Corresponding author. Tel.: +86 10 82801584; fax: +86 10 82802791.E-mail address: [email protected] (X. Rong Qi).

0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2006.11.006

only infects the hepatocytes rather than nonparenchymal cells inthe liver [8], the oriented delivery to hepatocytes would increasethe transfection efficiency and decrease the cytotoxicity of theasODN and the cationic liposomes theoretically. ASGPR-mediated endocytosis was known to be an efficient liver-tar-geting pathway. There are several galactose- or lactose-termi-nated compounds such as asialoorosomucoid [9], galactosylatedpoly-L-glutamic acid [10], asialofetuin glycopeptide [11],lactosylceramide [12] etc., to be used as the target ligands inthe liver-targeting liposomes, polymers or nanoparticles [13].Sito-G is the major component of soybean sterylglucoside (SG),a kind of plant extract mixture with all the componentscontaining one glucose residue [14]. Both SG and sito-G couldbe recognized specifically by ASGPR and the neutral liposomescontaining SG or sito-G could target the liver [14,15].Meanwhile, SG and sito-G are abundant and inexpensive,which was advantageous for them to be developed as a liver-

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282 Y. Zhang et al. / Journal of Controlled Release 117 (2007) 281–290

targeting ligand, because most of the other ligands mentionedabove were expensive to synthesize and purify. Hwang et al.reported the cationic liposomes containing sito-G as the liver-targeting gene delivery carrier and enhanced transfectionefficiency by sito-G [16]. We also reported that the cationicliposomes containing SG showed liver-targeting effect [17].Nonionic surfactants, for example, Tween 80, had improved thetransfection efficiency of cationic emulsions although themechanisms were unclear [18]. In the preliminary study, wecompared the transfection enhancement towards the cationicliposomes by the different modifications with several nonionicsurfactants such as Tween 80, Span 80, Brij 35 etc., and Brij 35showed to be the most efficient one.

In this study, we designed the novel cationic liposomes co-modified with sito-G and Brij 35, to enhance the delivery of a15-mer anti-HBV asODN orientedly to the hepatocytes, andevaluated the transfection efficiency and the antigen inhibitioneffect (i.e. inhibitory effect of asODN to virus antigensproduction) of the encapsulated asODN in vitro. For thesuccessful design and optimization of cationic liposomes, thereare many intricate steps to be considered for the cellular uptakeof asODN mediated by the cationic liposomes [4,19]. In thisstudy, with the help of some inhibitors that could act on differentsteps of the cellular uptake, such as asialofetuin, wortmanninand nigericin, the cellular uptake mechanisms and intracellulardistributions of both cationic liposomes and encapsulatedasODN were analyzed based on the antigens inhibition effect,FCM and CLSM.

2. Materials and methods

2.1. Materials

Dipalmitoyl-phosphatidylcholine (DPPC) was supplied byNOF Corporation (Tokyo, Japan). Dioleoyl-phosphatidyletha-nolamine (DOPE) was purchased from Northern Lipids(Vancouver, Canada) and rhodamine-DOPE was synthesizedby our laboratory. 3β[N-(N′,N′-dimethylaminoethane)-carba-moyl] cholesterol (DC-Chol) was synthesized by our laboratory[20]. Sito-G was isolated and purified from the SG mixture byHPLC in our laboratory. Brij 35 was purchased from Serva(Heidelberg, Germany). Asialofetuin (type I from fetal calfserum), wortmannin and nigericin were purchased from Sigma-Aldrich (St. Louis, USA). Dulbecco's modified Eggle'smedium (DMEM) and William's E medium were purchasedfrom Gibco BRL/Life Technologies (New York, NY, USA).Fetal bovine serum (FBS) was purchased from Tianjin CaihuiTechnologies (Tianjin, China). HepG2.2.15 cells were providedby Hepatology Institute, Peking University People's Hospital(Beijing, China). Male Wistar rats with the body weight 150 g–200 g were purchased from Laboratory Animal Center, PekingUniversity Health Science Center.

2.2. Cells culture

The HepG2.2.15 cells, a human hepatoblastoma cell linewhich was integrated with the HBV DNA into the chromo-

some and could stably produce HBV in the culture system,were cultured in WPS-DMEM (DMEM containing 10% (v/v)heat-inactivated FBS, 1% (w/v) penicillin and streptomycin) inthe humidified atmosphere with 5% CO2 at 37 °C. For thetreatment with cationic liposomes, the cells at not more thanpassage 20 were plated with the density of about 4×104/cm2

and then incubated for 1 or 2 days till the cell confluencereached at least 70%.

Primary rat hepatocytes were isolated and cultured based onthe Seglen two-steps method with some modifications [21]. Theprepared hepatocytes were cultured in the MWEM (themodified William's E medium).

2.3. Oligonucleotides

The 15-mer phosphorothioate oligonucleotides (asODN)which had the anti-HBV sequence 5′>GAT GAC TGT CTCTTA<3′, aimed at cap site/SP II of the HBV mRNA, wassynthesized on a solid phase DNA synthesizer (PE-ABI 391EP,USA) by phosphite-triester method and was purified by SDS-PAGE. For the FCM and CLSM analysis, the asODN was stilllabelled on the 5′ end with fluorescin isothiocyanate (FITC).

2.4. Preparation of cationic liposomes

Cationic liposomes encapsulating asODN or FITC-asODNwere prepared by film dispersion method as described in theprevious study [17]. The liposomes, CL (DPPC:DC-Chol=20:10), CL/sito-G (DPPC:DC-Chol:sito-G=20:10:1.34), CL/Brij 35 (DPPC:DC-Chol:Brij 35=20:10:1.34), and CL/Brij 35/sito-G (DPPC:DC-Chol:Brij 35:sito-G=20:10:1.34:1.34) wereprepared respectively (all the ratios in the formulations abovewere molar ratios). The stock concentration of asODN or FITC-asODN and the total lipids in the cationic liposome suspensionwere 28.8 μMand 0.9mg/mL respectively. Before administratedto the cells, the liposomeswere diluted byNPS-DMEM (DMEMcontaining 10% serum and no penicillin–streptomycin) to thefinal concentration, expressed as the concentration of theencapsulated asODN. The particle size, zeta potential, andpolydispersion index of CL/Brij 35/sito-G were about 155 nm,30.0 mV and 0.191, respectively, determined by Zeta-Sizer(Malvern, UK).

2.5. Flow cytometry analysis

Primary rat hepatocytes were prepared and plated onto the24-well plate with the density of 2×105 cell/well. Afterincubation for 2 or 3 days when the cells were growing well,the cells were washed once with MWEM, followed by theaddition of the cationic liposomes encapsulating FITC-asODNor free FITC-asODN solution at each given concentration. Afterincubation for the scheduled time, the cells were detached fromthe culture plate with 4% (w/v) trypsinase solution and washedthree times with PBS by centrifuging at 1000 r/min for 5 min.Finally the cells were suspended in PBS and fixed with theFCM fix solution (containing formalin 1 mL, glucose 2 g,sodium azide 0.2 g, PBS to 100 mL). Then the cell suspension

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(more than 10000 cells) was stored at 4 °C for FCM analysis(Beckton Dickinson, Belgium).

2.6. Determination of HBsAg and HBeAg production

HepG2.2.15 cells were plated. The asODN-encapsulatingcationic liposomes or the free asODN solution was added withthe given concentrations. The treatment agents were refreshedevery 24 h for 3 days with the unchanged concentration. Thesolutions dropped out from the culture wells were collected forthe HBsAg (hepatitis B surface antigen) and HBeAg (hepatitisB e antigen) determination. HBsAg and HBeAg productionswere determined by ELISA using Akzo Nobel HBsAg ELISAkit (Biomerius, France) and Radim HBeAg ELISATest System(Radim, Italy), respectively. All the results were expressed asthe optical density at 450 nm [OD (450 nm)].

In order to investigate the cellular uptake mechanisms ofthe encapsulated asODN in CL/Brij 35/sito-G, the inhibitorslike asialofetuin, wortmannin and nigericin were added intothe medium with the final concentrations 1.0 mg/mL, 0.1 μMand 1.0 μM, respectively, 10 min before the treatment withthe asODN-encapsulating cationic liposomes. Then theHBsAg and HBeAg productions were determined as de-scribed above.

2.7. Cytotoxicity assay

The cytotoxicity of the modified cationic liposomesencapsulating asODN was determined by MTT assay. Thecationic liposomes encapsulating asODN were added at thegiven concentrations. After incubation for 72 h withoutrefreshing the medium, 20 μL MTT solution (5 g/L) dissolvedwith PBS buffer (pH 7.2) was added in and then the cells wereincubated for 4 h to allow the production of the dark-blueformazan crystal. Then 200 μL of dimethylsulfoxide was addedinto each well. When the formazan crystal was dissolvedcompletely, the optical density at 490 nm [OD (490 nm)] wasdetermined by a microplate reader. The viability of the treatedcells was defined as follow:

The cell viability=OD (490 nm) of the treated cells /OD(490 nm) of the non-treated cells.

2.8. Fluorescent microscopy and confocal laser scanningmicroscopy analysis

Primary rat hepatocytes were prepared and plated with thedensity of 4×105 cell/well onto the 6-well plate with a coverglass (22 mm×26 mm) in each well. After incubation for 2 or3 days, the cells were washed once with MWEM, followed bythe addition of the cationic liposomes containing rhodamine-DOPE (1%, molar ratio in liposome formulation) andencapsulating FITC-asODN at the given concentrations. Afterincubation for the scheduled time, 5 μL Hoechst 33258 (1 mg/mL) was added into the medium to stain the nuclear for 30 min.Then the cells on the cover glass were washed for 3 times withPBS, fixed with 4% paraformaldehyde (dissolved with PBS) for15 min, and then stored at −20 °C for fluorescence microscopy

(Olympus, Japan) or confocal laser scanning microscopy(Leica, Germany) analysis.

3. Results

3.1. Transfection efficiency of asODN

To investigate the transfection efficiency of cationic lipo-somes in primary rat hepatocytes, the cellular uptake of theFITC-asODNwas evaluated by FCM.When treated for 6 h withfree FITC-asODN solution at 0.36 μM, the primary rathepatocytes exhibited 10.9% transfection efficiency. Comparedto free FITC-asODN solution, the modified cationic liposomesencapsulating FITC-asODN (CL/sito-G, CL/Brij 35 and CL/Brij 35/sito-G) increased transfection efficiency by 4.4- 7.1folds, but the unmodified cationic liposomes (CL) increasedonly 1.0 fold (transfection efficiency of 21.9%). In the case ofCL/Brij 35 and CL/sito-G, the transfection efficiency values(60.0% and 59.0%) were similar to each other. CL/Brij 35/sito-G, the liver-targeting cationic liposomes co-modified with sito-G and Brij 35, exhibited the strongest transfection efficiency(88.7%). The mean fluorescence intensity (MFI) of CL/Brij 35/sito-G reached the maximum at 6 h and the MFI value at 6 h wasalmost as much as that at 24 h (Fig. 1a), indicating that thecellular uptake of asODN was time-dependent at the initialperiod of 6 h and then maintained nearly a constant.Furthermore, from the data with 6 h treatment at variousFITC-asODN concentrations, the cellular uptake of asODN wasconcentration-dependant, and when the FITC-asODN concen-tration increased to 1.80 μM, the transfection efficiency reachedalmost 100% (Fig. 1b).

3.2. Biological effect on HBV antigens production

HBsAg and HBeAg, two antigens produced with theprogress of HBV infection, are diagnosis indicators of chronichepatitis B. When the HBsAg or HBeAg level is up or down,the corresponding optical density [OD (450 nm)] will increaseor decrease in the antigen production determination, so theantigen level (HBsAg or HBeAg production) can be expressedas OD (450 nm). Here we defined the term “antigen inhibitioneffect” to describe the inhibitory effect of asODN to HBVantigen production level, which represented the biological effecton HBV antigens production. As shown in Fig. 2, after 3 daystreatment with 3.6 μM asODN, CL/Brij 35/sito-G brought themost reduction of antigens production (both HBsAg andHBeAg) of HepG2.2.15 cells and therefore showed thestrongest antigen inhibition effects. CL/Brij 35 and CL/sito-Gbrought more reduction in HBsAg production than CL (Fig. 2a).As for the HBeAg production (Fig. 2b), compared with notreatment group and free asODN solution, both CL/Brij 35 andCL/Brij 35/sito-G showed significant inhibition on HBeAgproduction, while neither CL nor CL/sito-G did. Although thetransfection efficiencies of CL/Brij 35 and CL/sito-G weresimilar to each other, the antigens inhibition effect of the formerwas stronger than that of the latter. The difference in the HBsAgand HBeAg production might respect to the complementary

Fig. 2. HBsAg (a) and HBeAg (b) production of HepG2.2.15 cells treated withdifferently modified cationic liposomes encapsulating asODN and free asODNsolution respectively for 3 days. The concentration of asODN was 3.6 μM. OD(450 nm) indicated HBsAg and HBeAg levels. Each value represented the mean±S.D. (n=3). ⁎P<0.05, ⁎⁎P<0.01, significantly different.

Fig. 1. Transfection activity at (a) different treatment time with 0.36 μM FITC-asODN and (b) different FITC-asODN concentrations with 6 h treatment whentreated with FITC-asODN-encapsulating CL/Brij 35/sito-G by flow cytometry.Here TE (bar) referred to transfection efficiency (%), defined as the percentageof the positive cells whose fluorescence intensity was more than 10 intensityunits; MFI (line) referred to mean fluorescence intensity (fluorescence units),defined as the arithmetical mean of the fluorescence intensity of all the countedcells. Values represented as mean of two independent experiments.

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sequence of the used asODN. The complementary sequence ofthis asODN was located in the cap site of 3.5 kb mRNA(involved in the transcription of HBsAg) and the middle site of2.4 kb mRNA (involved in the transcription of HBeAg) at thesame time [8]. It was easier for the cap site to be bound byasODN than the middle site, so HBsAg level was inhibited morethan HBeAg level, i.e., the HBsAg inhibition would be moresignificant.

The relationship between the antigens production inhibitionto HepG2.2.15 cells and the concentration of the asODNencapsulated in CL/Brij 35 /sito-G was determined. In the rangeof 0.18–7.2 μM, the HBsAg production [OD (450 nm)]-concentration curve was an exponential one: (OD (450 nm)=−0.187 ·LnCasODN+0.445, r2 =0.987) (Fig. 3a), and theHBeAg production [OD (450 nm)]-concentration curve wasin shape of counter-S (Fig. 3b): OD (450 nm)= (146.3+0.44 ·CasODN

6.7 ) / (146.3+CasODN6.7 ) (r2 =0.955). These findings

suggested that the HBsAg and HBeAg inhibition effects wereconcentration-dependant, but after the asODN concentrationincreased to a certain level, the inhibition effects were strength-ened little with the increase of the asODN concentration.

Fig. 3. Relationship between HBsAg (a) and HBeAg (b) production andconcentration of asODN encapsulated in CL/Brij 35/sito-G. HepG2.2.15 cellswere treated with the cationic liposomes at the asODN concentration from 0 to7.2 μM for 3 days. In both figures, OD (450 nm) indicated HBsAg and HBeAglevel. The line meant the estimation curve: a. HBsAg, OD(450 nm)=−0.187 ·LnCasODN+0.445, r2=0.987); b. HBeAg, OD(450 nm)= (146.3+0.44·CasODN

6.7 ) / (146.3+CasODN6.7 ) (r2=0.955). Each value represented the mean±

S.D. (n=3).

Fig. 5. Series of CLSM section images of the primary rat hepatocyte treated withCL/Brij 35/sito-G encapsulating FITC-asODN. The CL/Brij 35/sito-G waslabelled with rhodamine by adding 1.0% (molar ratio) rhodamine-DOPE in theliposome formulation. The concentration of FITC-asODN was 1.8 μM. Aftertreatment for 6 h, Hoechst 33258 was added into the medium to stain the nuclearfor 30 min. The green, red, and blue fluorescence indicated FITC-asODN, thelipids, and the nuclear, respectively. The three groups of A, B and C wereobtained from different excitation wavelengths. Group A showed the nuclear,group B showed the asODN, and group C showed the rhodamine labelledcationic liposomes. Group D was obtained by overlaying group A, B and Cthrough the image process system of CLSM, and showed the overallfluorescence characteristics of the cell. (For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of thisarticle.)

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3.3. Cytotoxicity

The cytotoxicities of CL and CL/Brij 35/sito-G encapsulat-ing asODN at the concentrations from 0 to 9 μM (asODN) weredetermined by MTT assay, respectively. As shown in Fig. 4, thecytotoxicities of both cationic liposomes were increased withthe elevation of asODN concentration, particularly in the highconcentration range from 3.6 to 9.0 μM. The increasedcytotoxicity may be caused by the cationic lipid DC-Chol,since the DC-Chol concentration was elevated accompaniedwith the elevation of asODN concentration. However, there wasno significant difference in the cell viability between CL andCL/Brij 35/sito-G group in the tested concentration range. Thisindicated that the co-modification of Brij 35 and sito-G broughtlittle influence to the cytotoxicity of cationic liposomes.

3.4. Cellular uptake and intracellular distribution in fluores-cence microscopy and CLSM

After treated with CL/Brij 35/sito-G encapsulating 1.8 μM ofFITC-asODN, the cells were observed through the fluorescencemicroscopy. Consistent with the results of transfection efficien-cy (Fig. 1) and antigens production (Fig. 2), most cells showedstrong fluorescence intensity (data not shown), indicating thatCL/Brij 35/sito-G led to high transfection efficiency. Besides, itcould be observed that the cell-associated fluorescence intensityincreased within the time period during 0 to 6 h and thefluorescence intensity at 24 h was similar to that at 6 h (data notshown).

In CLSM, FITC-asODN and rhodamine-DOPE for lipo-somes were used at the same time to serve as the fluorescenceprobes. In the CLSM images, the green, red and bluefluorescence indicated FITC-asODN, lipids and the nuclear,respectively. A series of CLSM section images (labelled as 1,2,… and 8 from top to bottom of the cell in order) wereobtained by scanning the primary rat hepatocytes at an interval

Fig. 4. Viability of HepG2.2.15 cells treated with CL and CL/Brij 35/sito-G atthe concentrations from 0 to 9 μM of asODN for 3 days. The cell viability wasdetermined by MTT assay. Each value represented the mean±S.D. (n=3).

of 1 μm thickness (Fig. 5). As shown in Fig. 5B, there wascontinuous green fluorescence in the nuclear which meant thatthe most amount of the asODN was distributed or accumulatedin the nuclear lumen. The continuous red fluorescenceappeared in the cytoplasm but not in the nuclear (Fig. 5C),and this might indicate the lipids or liposomes did not enter thenuclear lumen after penetrating into the cell. In the cytoplasm,especially near the nuclear, the punctuate green and redfluorescence appeared at the same place (Fig. 5D). In sectionB4–B6, a few punctuate fluorescence particles appeared on thecell membrane just like those in the cytoplasm, indicatingsome of the asODN-encapsulating liposomes was endocytosedto form endosomes. These fluorescence particles in thecytoplasm seemed to be the endosomes that had not yetreleased the asODN.

3.5. Mechanisms of the cellular uptake of CL/Brij 35/sito-G

The cellular uptake and delivery mechanisms of theencapsulated asODN in CL/Brij 35/sito-G were studied byevaluating the HBsAg inhibition effect of asODN in ELISA andby observing its intracellular distribution in CLSM.

In the presence of asialofetuin, the HBsAg inhibition effectsof CL/sito-G and CL/Brij 35/sito-G were reduced, while that of

Fig. 6. Effect of asialofetuin on HBsAg inhibition effect of the asODNencapsulated in the cationic liposomes. HepG2.2.15 cells were treated with thecationic liposomes encapsulating asODN (3.6 μM) for 3 days, in the presence orabsence of asialofetuin (1 mg/ml). The HBsAg production was determined byELISA. Each value represented the mean±S.D.(n=3). ⁎⁎P<0.01, significantlydifferent.

Fig. 7. Effect of wortmannin and nigericin on the antigens inhibition effect of theasODN encapsulated in CL/Brij 35/sito-G. HepG2.2.15 cells were treated withCL/Brij 35/sito-G encapsulating asODN (3.6 μM) for 3 days, in the presence orabsence of wortmannin (0.1 μM) or nigericin (1.0 μM). The antigens productionwas determined by ELISA. Each value represented the mean±S.D. (n=3).⁎⁎P<0.01, significantly different.

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CL was not (Fig. 6). As the result, the inhibition effects of CL/sito-G and CL reached almost the same level. Asialofetuindecreased the HBsAg inhibition effects of the sito-G containedcationic liposomes (i.e. CL/sito-G and CL/Brij 35/sito-G) butaffected little on the non-sito-G contained cationic liposomes(i.e. CL), because the recognition of ASGPR to sito-G could beblocked when the sito-G contained cationic liposomes wereco-incubated with 1.0 mg/mL asialofetuin which could berecognized by ASGPR specifically. However there was nostatistically significant difference between “with” and “with-out” asialofetuin for the HBsAg inhibition effect of CL/Brij 35/sito-G, in which Brij 35 seemed to decrease the specificrecognition of ASGPR to sito-G in CL/Brij 35/sito-G. Thephenomenon could be explained according to the followings:(1) Both Brij 35 and sito-G have amphiphilic structure andcould insert into the bilayer membrane of liposomes. Since Brij35 has a long PEG chain that could extend outside and bringsteric hindrance [22], it might prevent part of ligands frombinding with the surface receptor and thus decrease thespecificity of CL/Brij 35/sito-G to hepatocytes; (2) Brij 35compensated the transfection reduction induced by asialofe-tuin, because both sito-G and Brij 35 could enhance thetransfection via different mechanisms, such as ASGPRpathway for sito-G and facilitating membrane fusion for Brij35. Consequently when the effect of sito-G was blocked, thetransfection reduction was not so much because of thetransfection enhancement of Brij 35 through facilitatingmembrane fusion.

Wortmannin is known as an inhibitor of endocytosis [23].When the cells were co-incubated with wortmannin, theantigens inhibition effect of CL/Brij 35/sito-G was drasticallydecreased by 70.7% for HBsAg (Fig. 7a) and 50.1% for HBeAg(Fig. 7b), compared with that of absence wortmannin group.This indicated that endocytosis was one of the uptake pathwaysfor the encapsulated asODN.

Nigericin is known as an antibiotic that can dissipate thepH gradient across the endosome membrane and thus inhibit

the delivery of asODN from the endosomes via membranefusion [23]. When the cells were co-incubated withnigericin, the antigens inhibition effect of CL/Brij 35/sito-G was sharply decreased by 44.1% for HBsAg (Fig. 7a)and 46.2% for HBeAg (Fig. 7b). This suggested thatnigericin inhibited the delivery of CL/Brij 35/sito-G fromendosomes via membrane fusion between liposome andendosome membrane.

Fig. 8 showed us the CLSM images of the primary rathepatocytes treated with CL/Brij 35/sito-G (Fig. 8a), and withCL/Brij 35/sito-G in the presence of wortmannin (Fig. 8b),nigericin (Fig. 8c) and asialofetuin (Fig. 8d), respectively. Inthe presence of wortmannin, there were lots of punctuatefluorescence particles (red and green) on the cell membranebut few or no in the cytoplasm, and only weak continuousfluorescence (red and green) in the cytoplasm and the nuclear.This showed us that wortmannin, an endocytosis inhibitor,inhibited the cellular uptake of CL/Brij 35/sito-G viaendocytosis. In the presence of nigericin, there were morepunctuate particles (red and green) in the cytoplasm than inthe absence of nigericin, but very weak continuous fluores-cence (red and green) in the cytoplasm and nuclear. Apossible explanation was that after the liposomes entered thecells via endocytosis to form endosomes, nigericin inhibitedthe delivery of asODN from liposomes via membrane fusionbetween endosomes and liposomes. In the presence ofasialofetuin, the cell showed similar fluorescence distributionbut weaker fluorescence intensity compared with that in theabsence of asialofetuin. This suggested that when theASGPR-mediated endocytosis was blocked competitively byasialofetuin, the transmembrane ability of CL/Brij 35/sito-Gwas reduced, which was consistent with the previous results,i.e., the antisense effect of CL/Brij 35/sito-G in the absence of

Fig. 8. CLSM images of the primary rat hepatocyte treated with CL/Brij 35/sito-G encapsulating FITC-asODN (1.8 μM). The CL/Brij 35/sito-G was labelled withrhodamine by adding 1.0% (molar ratio) rhodamine-DOPE in liposome formulations. The concentration of FITC-asODN was 1.8 μM. The inhibitors such asasialofetuin, wortmannin and nigericin were added into the medium with the final concentrations 1.0 g/ml, 0.1 μM and 1.0 μM, respectively. After treatment for 6 h,Hoechst 33258 was added into the medium to stain the nuclear. The green fluorescence indicated FITC-asODN, the red indicated the lipids and the blue indicated thenuclear. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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asialofetuin was higher than that in the presence ofasialofetuin (Fig. 6).

4. Discussion

In this study, the novel cationic liposomes co-modified withligand and nonionic surfactant, was developed as a gene transfercarrier to deliver an anti-HBV asODN orientedly to thehepatocytes. Although the cellular uptake of asODN wasdependant on cell types, cell culture conditions, media,sequences, length of the sequences etc., people usually took itfor granted that gene (oligonucleotides and plasmids) could notenter the cells easily because of the strong electrostatic repulsionbetween the nucleotides chain and the cell membrane. Our

results showed that free asODN could be taken up byhepatocytes even at the relatively low concentration(0.36 μM), despite insufficient antigens inhibition effectcompared with the effect of the asODN encapsulated in cationicliposomes. The possible reason is the inherent strong uptakeability of hepatocytes, based on the large cell size and biologicalrequirements for the strong metabolic ability of the liver. Similarto our results, Robaczewska et al. reported strong fluorescenceintensity of the hepatocytes when injected with free FITC-asODN solution [24] and Soni et al. reported more efficient virusinhibition of free asODN solution than that of unmodified neutralasODN-encapsulating liposomes when injected to ducklingsinfected with duck HBV [25]. With the co-modification of Brij35 and sito-G, CL/Brij 35/sito-G showed not only high efficient

Fig. 9. A schematic representation shows the cellular uptake and delivery ofasODN from CL/brij35/sito-G in hepatocytes. Endocytosis pathway: theliposomes were taken up by the membrane endocytosis with endosomes formedand then asODN was released by membrane fusion between the endosomes andthe liposomes. ASGPR pathway: the liposomes were taken up based on thespecific recognition of the ligand sito-G by ASGPR and the endosome wasformed. Membrane fusion pathway: asODN was released into the cytoplasm atthe membrane by the fusion between cell membrane and liposome membrane.After being released, most of the asODN would be accumulated into the nuclear.

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transfection and antigens inhibition but hepatocyte-targetingeffect based on the receptor binding competition test, and the co-modification did not increase cytotoxicity to the hepatocytes,even when the cells were treated with the cationic liposomes foras long as 3 days. Phosphorothioate oligonucleotides are knownto be resistant to nuclease degradation, but not resistant toRNaseH degradation [26]. When the concentration of asODN inthe cytoplasm was elevated to a certain level, there might berelatively more RNaseH to be activated quickly, and thereforethe degradation of the asODN was accelerated. Besides,HepG2.2.15 is a cell line that can stably produce HBVmRNAs and antigens. In spite of the treatment with asODN,the HBV DNA integrated into the cell chromosome could stilltranscript more and more mRNAs all the time to make up for thedegradation of the mRNA, so the virus or the virus antigenscould not be cleared completely by the asODN, which was whythe antigens inhibition was not complete.

Through CLSM, people could clearly observe the distribu-tion of the fluorescence probes in the cells and get some detailedinformation about the intracellular behaviors of the liposomesand the asODN. It was known that when microinjected to thecytoplasm, free FITC-asODN could substantially accumulate inthe nuclear rapidly by passive diffusion through the membrane.Interestingly, we found that asODN encapsulated in the co-modified cationic liposomes accumulate in the nuclear aftertransfer into the cells (Fig. 5). One of the possible reasons wasthat the asODN was recruited and immobilized by theinteraction between asODN and the positively charged intra-nuclear components such as histones [27]. Another reason wasthat the asODN might be associated with the intracellular RNAmatrix [28]. As for the antisense therapy, the accumulation ofasODN in the nuclear was advantageous, because mRNA,which was aimed at by asODN, would be transcripted in thenuclear first and then diffuse out to the cytoplasm. In Figs. 5 and8, there were many fluorescent particles near the nuclear or onthe nuclear membrane, which suggested that maybe theendosomes were associated with the nuclear membrane andthen the asODN would be delivered based on the membraneinteraction. Shi et al. reported that the cationic liposomes–asODN complex was released from the artificially rupturedendosomes with the dissociation of cationic lipids and asODNat the nuclear membrane [28]. Some cationic polymers such aspolyethylinemine (PEI) could mediate the entry of plasmidDNA into the nuclear at the nuclear membrane [29].

The ASGPR-mediated targeted gene carriers were developedby many groups, such as cationic liposomes, polymers, andrecombinant lipoproteins, for the gene to be delivered to theliver, especially to hepatocytes. Since HBV only replicates inhepatocytes but does not in other cells, it is required to confirmthe hepatocyte specificity of ligand in the liver-targeting genedelivery carrier. We already reported that the distribution of SGand Brij 35 co-modified liposomes in rat hepatocytes (contain-ing ASGPR on the cell surface) were higher than in non-hepatocytes (containing no ASGPR), and the transfection inHepG2.2.15 cells could be inhibited by pretreating the cellswith galactose [30]. Also, the distribution of 3H-labelled SGcationic liposomes in rat hepatocytes was higher than that of

non-SG liposomes [17]. Furthermore, transfection of SG-liposomes as gene delivery carrier in HepG2 cells was moreefficient than in HeLa human cervix carcinoma cells, 293human kidney epithelial cells and Y1 mouse hormone-secretingadrenal cortex cells [31]. Hwang et al. suggested that thecationic liposomes containing sito-G on the liposomes surfacebrought significant improvement to the transfection efficiencyin HepG2 cells and attributed this improvement to the ASGPRrecognition to sito-G [16], but provided no direct evidenceabout receptor binding test. Asialofetuin or galactosylatedalbumin were often used to evaluate the hepatocyte-targetingeffect via ASGPR in vitro or in vivo, because they could bind toASGPR specifically and thus block the receptor completely. Inthe presence of asialofetuin or galactosylated albumin thehepatocyte-targeting effect would be inhibited completely andthe transfection efficiency would also be decreased [32–34]. Inthis study, the modification with sito-G in the cationicliposomes (CL/sito-G and CL/Brij 35/sito-G) enhanced thetransfection efficiency and improved the antigens inhibitioneffect in vitro. Moreover, the receptor binding competition testwith asialofetuin suggested that the improvement was depen-dent on the ASGPR-mediated pathway, which meant that sito-Gwould be a useful ligand in the ASGPR-mediated hepatocyte-targeting.

Endocytosis and membrane fusion were the major pathwaysof the asODN cellular uptake, and the former was usually theprimary step in the efficient gene transfection. Noguchi et al.suggested that either wortmannin (endocytosis) or nigericin(membrane fusion) might completely inhibit the asODNtransfection, because membrane fusion was the subsequentstep upon endocytosis [35]. In our experiments, as for the co-modified cationic liposomes, both wortmannin and nigericinpartly inhibited the asODN transfection, which suggested thatthere might be some cellular uptake pathways for the co-modified cationic liposomes other than endocytosis and

GENEDELIVERY


membrane fusion in endosomes. For example, fusion might alsooccur between liposomes and cell membrane and the asODNwould be released across the cell membrane, because Brij 35could destabilize the cell membrane as the nonionic surfactantand thus induce the lipid exchange between liposomes and cellmembrane [18]. Different lipids and cell types could result indifferent cellular uptake processes of the cationic liposomes, asreviewed by Duzgune and Nir [19]. As reported previously,phosphatidylcholine (PC) like DPPC was generally inhibitoryto membrane fusion while phosphatidylethanolamine (PE) likeDOPE could facilitate it, and consequently, PE-cationicliposomes showed more efficient transfection than PC-cationicliposomes [19]. In this study, although containing DPPC as co-lipid, the cationic liposomes resulted in membrane fusion inendosomes. Considering that Brij 35 has a long oxyethylenechain which would destabilize the endosome membrane [18]and a long alkyl chain which might improve the fluidity ofcationic liposomes, it could be presumed that it was the additionof Brij 35 that improved the transmembrane ability of thecationic liposomes by fusion process. Kim et al. also reportedthat Tween 80 could enhance “lipofection” of the emulsion–DNA complexes, and presumed that Tween 80 had thefusogenic property [18]. Besides, sito-G might also contributepartly to the membrane fusion because of its penetration-enhancing effect [16].

With the help of several inhibitors acting on different stepsfor the encapsulated asODN to be delivered into the cells, thecellular uptake mechanisms of the encapsulated asODN in CL/Brij 35/sito-G (Fig. 9) were concluded to be a complicatedprocess which involved at least endocytosis, membrane fusionand ASGPR-mediated pathway (also endocytosis). Sito-Gshowed to be a useful ligand in the liver-targeting genedelivery, and Brij 35 seemed to be able to facilitate themembrane fusion in the lipofection. Based on this conclusion, itmight be most meaningful for combining the ligand with thenonionic surfactant in the oriented delivery of asODN tooptimize the transfection in vivo, because the enhancedendocytosis by the ligand, and the facilitated delivery fromthe endosomes by the nonionic surfactant, would be achieved atthe same time. If necessary, some bio-surfactants that may besafer could be used to substitute Brij 35 in vivo.

Acknowledgement

This work was supported by the National Natural ScienceFoundation of China, 30371265 and 90406024.

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