Gene delivery using liposome technology

Journal of Controlled Release 62 (1999) 269–277www.elsevier.com/ locate / jconrel

Gene delivery using liposome technologya , a a a*Hiroshi Kikuchi , Norio Suzuki , Kiyoslii Ebihara , Hiromi Morita ,

b c c cYoshikazu Ishii , Akira Kikuchi , Susumu Sugaya , Takehiro Serikawa ,cKenichi Tanaka

aPharmaceutical Formulation Research Laboratory, Daiichi Pharmaceutical Co., Ltd, Edogawa-ku, Tokyo 134-8630, JapanbExperimental Technology Research Center, Daiichi Pharmaceutical Co., Ltd, Edogawa-ku, Tokyo 134-8630, Japan

cDepartment of Obstetrics and Gynecology, Niigata University School of Medicine, Asahimachi, Niigata 951-8510, Japan

Abstract

Development of more reliable liposomal formulations and preparation methods which can be used for gene therapy insteadof commonly used viral vectors is expected. We have already developed the freeze-dried empty (non-drug-containing)liposomes (FDEL) method for mass-production of liposomal products. After these freeze-dried empty liposomes arerehydrated with aqueous drug solutions, many kinds of drugs can be encapsulated highly efficiently, and particle size can becontrolled well. This study evaluated the usefulness of this FDEL method for preparation of liposomes containing DNA witha particular attention to the stability of DNA. When the liposomes were prepared by the conventional lipid-film method on arelatively large scale with use of a Potter-homogenizer (a teflon homogenizer), significant degradation and conformationalchange of DNA was observed during homogenization. Loss of DNA was also significant after extrusion for sizing andsterilization; residual DNA in the final preparation was hardly detected. When the FDEL method was used, on the otherhand, no degradation, conformational change or loss of DNA was observed, and particle size was easily controlled.Moreover, there was no significant difference in luciferase activity between the lipid-film method used on a small scale withuse of a vortex mixer and the FDEL method after transfection of tumor cells (HRA, HEC-1A and Colo320DM) by theliposomes containing DNA (PGV-C). These findings suggest that the FDEL method is very useful for preparation ofliposomes containing DNA. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cationic liposomes; Freeze-dried empty liposomes (FDEL); Plasmid DNA; Stability; Transfection

1. Introduction adeno-associated viruses [3], etc. Although viralvectors yield efficient gene transfer both in vitro and

More than 2000 patients in the world have been in vivo, there are major limitations associated withalready treated with gene therapy since 1990, using this approach: immune response against the transfec-viral vectors and non-viral vectors. The viral vectors tion systems, limits on the size of the plasmid to bein general use are retroviruses [1], adenoviruses [2], incorporated, the possibility of recombination, and

difficulties in scaling up vector production [4]. Viralvectors are mainly used for ex vivo gene therapy*Corresponding author. Tel.: 181-3-5696-8303; fax: 181-3-because of safety problems.5696-8228.

E-mail address: [email protected] (H. Kikuchi) As non-viral vectors, polycations (polylysine

0168-3659/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 99 )00047-4

270 H. Kikuchi et al. / Journal of Controlled Release 62 (1999) 269 –277

[5,6], polyethyleneimine [7]) and particulate carriers(liposomes [8,9], nanoparticles [10]) have beenstudied. Polycations and particulate carriers offerseveral advantages over most commonly used viralvectors: they are much safer and simpler to use andeven less expensive to produce. In addition, in thecase of liposomes, it is easier to modify the liposom-al surface with sugars such as galactose [11,12] andmannose [13], or with hydrophilic compounds suchas polyethyleneglycol [14] and polyglycerols [15],for targeting liposomes to specific tissues or cells.Liposomes are thus expected to be useful for in vivogene therapy. However, commercialized cationic Fig. 1. Procedures for preparation of liposomes containing drug:liposomes have some disadvantages [16]: (1) they (a) the lipid-film method is the conventional one which is

commonly used; (b) the freeze-dried empty liposomes (FDEL)are not so stable after purchase, during storage andmethod is a newly-developed one which does not require heat andafter mixing with the DNA solution; (2) they areshear stress even after an aqueous drug solution is added.strongly influenced by the pH and/or osmolarity of

the added DNA solution; (3) most of them are notavailable, except for some formulations such as

RGenetransfer , for use in the presence of fetal bovine drug can be obtained. It appears that the highserum (FBS) due to alterations of their vesicle temperature and shear stress used in the abovestructures resulting from interaction with serum process negatively affect the stability of relativelycomponents. This implies these cationic liposomes labile drugs such as proteins and DNAs. In the caseare not useful for in vivo gene therapy. Development of the FDEL method, after water is added to theof more reliable liposomal formulations and prepara- lipid-film, procedures such as hydration, emulsifica-tion methods is needed, and, in particular, DNA must tion, sizing and filtration are carried out above Tc ofbe stable in the liposomal formulations and in body the lipids, and the dispersion of the empty liposomesfluids. It is very unfortunate, however, that there not containing the drug is obtained. Next, a definitehave been few systematic studies solely devoted to amount of these empty liposomes is pipetted intoexamination of the stability of DNA itself or the each glass vial, and through the following lyophiliza-DNA associated with liposomes. This study evalu- tion, liposomal membrane is seemed to be broken toated the usefulness of the freeze-dried empty lipo- pieces. Here, if an aqueous drug solution is added tosomes (FDEL) method [17,18] for preparation of the FDEL in the vial and the vial is shaken by handliposomes containing DNA with a particular atten- at room temperature, the liposomes containing drugtion to the stability of DNA. are formed very easily, as shown in Fig. 2. With this

simple procedure, many kinds of drugs can beencapsulated highly efficiently and the particle size

2. Principle of the FDEL method can be controlled well [17]. For example, in the case

Fig. 1 shows two kinds of preparation methods forliposomes containing a drug: (a) the lipid-film meth-od, and (b) the freeze-dried empty liposomes (FDEL)method. The lipid-film method is the conventionalpreparation method: after preparation of the lipid-film, an aqueous drug solution is added, and throughhydration, emulsification, sizing and filtration abovethe gel–liquid crystalline phase transition tempera- Fig. 2. Schematic diagram showing use of freeze-dried emptyture (Tc) of the lipids used, liposomes containing a liposomes (FDEL) to obtain liposomes containing drug.

H. Kikuchi et al. / Journal of Controlled Release 62 (1999) 269 –277 271

of doxorubicin, the efficiency of drug encapsulation plemented with 10% FBS until the transfectionis almost 100% and the particle size is 100–200 nm. experiment.

3.2. Stability of naked DNA

3. Materials and methodsThe stock solution of DNA (pbgal-Control) was

diluted to 50 mg/ml with a 9% sucrose aqueous3.1. Materials solution (pH 7.0), and the stability of naked DNA

against various procedures was investigated. First, inTwo plasmid vectors used in this study were order to examine whether DNA adheres to vessels, a

prepared using the QIAGEN Plasmid Mega kit polypropylene tube, a round-bottomed glass flask or(Qiagen, Hilden, Germany), resuspended in distilled a Potter-homogenizer (teflon pestle and glass vessel)water at about 1–2 mg/ml, and stocked at 2208C were used: 5 ml of the DNA solution was placed inuntil use. For the stability study, pbgal-Control the above vessel at 658C for 10 min. Second, the(Promega, Madison, MI), which is pbgal-basic with influence of emulsification was examined: 5 ml ofthe SV-40 promotor and enhancer, was used, and for the DNA solution in the glass vessel was emulsifiedthe luciferase activity study, PGV-C (Toyo Ink, by a bath-type sonicator, a vortex mixer, or a Potter-Tokyo, Japan), which has the coding sequence of the homogenizer at 658C for 10 min. Thirdly, thefirefly luciferase under the control of SV-40 promotor influence of the membrane filter was investigated: 5and enhancer, was used, respectively. It was con- ml of the DNA solution preheated at 658C wasfirmed by gel electrophoresis that each plasmid DNA filtered repeatedly in triplicate through PVDF mem-had one major band which corresponds to a closed- brane filter (pore size, 0.45 or 0.22 mm) or CEcircular (supercoiled) DNA. The molecular weight membrane filter (pore size, 0.22 mm). After theladder lHindIII was obtained from Takara Shuzo, above treatments, the residual DNA was character-Co., Ltd. (Osaka, Japan). ized by gel electrophoresis, and an Intelligent Quan-

N-[a-Trimethylammonioacetyl]-didodecyl-D-gluta- tifier (Biolmage Co., Ltd., MI, USA) was used formate chloride (TMAG) was purchased from Sogo determination of the quantity of the closed-circularPharmaceutical Co., Ltd. (Tokyo, Japan), and L-a- (supercoiled) DNA and the open-circular DNA.dioleoylphosphatidylethanolamine (DOPE) and L-a-dilauroylphosphatidylcholine (DLPC) were from 3.3. Preparation of liposomes by the FDELNOF Corporation (Tokyo, Japan). Poly- methodvinylidenedifluoride (PVDF) membrane filters (25mm f pore size, 0.45 and 0.22 mm) and cellulose A lipid composition of TMAG/DOPE/DLPC in aacetate /cellulose nitrate mixed esters (CE) mem- molar ratio of 2:4:4 was used in this study. Thebrane filters (25 mm f; pore size, 0.22 mm) were lipid-film of this composition is commercially avail-

Rpurchased from Nihon Millipore Ltd. (Tokyo, able as the reagent Genetransfer (Wako, Osaka,Japan). All other chemicals were of commercial Japan), and this reagent exhibits great transfectionanalytical grade and used without further purifica- activity even in the presence of FBS [19].tion. Vials of FDEL were prepared in a scale of 500 ml

The human ovarian cancer cell line HRA was (0.5 ml31000 vials) as follows: TMAG, DOPE andprovided by Dr. Y. Kikuchi (Department of Ob- DLPC in a molar ratio of 2:4:4 were dissolved instetrics and Gynecology, National Defense Medical chloroform, and the solvent was removed in a rotaryCollege, Japan). The endometrial cancer cell line evaporator. The lipid mixture was evacuated in aHEC-1A was purchased from American Type Cul- desiccator under reduced pressure for more than 12ture Collection, and the human colon cancer cell line h, and an exact amount of lipids was weighed into aColo320DM was obtained from the Japan Health 1000 ml beaker. To this lipid mixture a 9% sucroseScience Foundation. All of the cell lines were aqueous solution was then added to make a finalmaintained in DMEM (IBL, Fujioka, Japan) sup- volume of 500 ml and make the final lipid com-


position of TMAG/DOPE/DLPC50.4 mM:0.8 holding the vessel on a water bath at 60–708C, themM:0.8 mM. After hydration of the lipids by holding hydrated dispersion was homogenized by a Potter-the beaker on a water bath at 60–708C, the hydrated homogenizer for 10 min. In order to obtain a moredispersion was homogenized by a T.K. Homo Mixer, homogeneous particle size distribution, the disper-type M (Tokushu Kika Kogyo Co., Ltd., Osaka, sion was extruded twice at 60–708C through a PVDFJapan) for 10 min. In order to obtain a more membrane filter with 0.22 mm pore size. After thehomogeneous particle size distribution, the disper- above procedures for homogenization, the first andsion was extruded twice at 60–708C through a PVDF the second extrusion, a small portion (about 0.1 ml)membrane filter with 0.22 mm pore size. An exact of the liposomal dispersion was taken as a sample foramount of the dispersion was pipetted into 2 ml glass gel electrophoresis.vials (0.5 ml portion each), frozen at 2508C for 4 h, In the case of liposomes prepared by the FDELand then lyophilized in a freeze-dryer (The Virtis method, after rehydration of the FDEL by a DNACo., Inc., New York, USA). Finally, each vial (pbgal-Control) aqueous solution and hand-shaking,contained a lipid composition of TMAG/DOPE/ the obtained liposomes containing DNA were ap-DLPC5200 nmol:400 nmol:400 nmol, and was plied to gel electrophoresis.stored at 48C until use. The DNA was extracted from the liposomal

An aqueous solution of 20 mg of DNA/463 ml of dispersion as follows: after 100 ml of the liposomaldistilled water was added to the FDEL, and the vial dispersion was treated with 200 ml of phenol /chloro-was shaken by hand at room temperature in order to form (1:1, v /v), DNA in the water phase wasobtain the liposomes containing DNA (1 mg of precipitated by a solution of 20% polyethyleneglycolDNA/10 nmol of TMAG, cationic lipid). Here, the 6000/2.5 M NaCl on an ice bath, and was dissolvedfinal volume of the dispersion became 0.5 ml (40 mg in a Tris-EDTA buffer. After extraction of DNAof DNA/ml of the dispersion), and the dispersion from the liposomal dispersion, a definite amount ofbecame isotonic. the extracted DNA solution (50 ng of DNA as the

initial amount) was applied to gel electrophoresis,3.4. Stability of DNA during preparation of which was carried out in the conventional fashionliposomes using lHindIII as a molecular weight ladder.

As control liposomes for DNA stability testing3.5. Characterization of the liposomes

during preparation, liposomes containing DNA wereprepared by the lipid-film method in a scale of 15 ml

The fundamental physicochemical properties, in-as follows. The procedures were almost the same as

cluding pH, particle size and zeta-potential, of thefor the FDEL method, except that a DNA aqueous

liposomes prepared by the lipid-film method and thesolution instead of a sucrose solution was added to

FDEL method were determined. The particle sizethe lipid mixture (lipid-film). Thus, TMAG, DOPE

and distribution of the liposomes were determinedand DLPC in a molar ratio of 2:4:4 were dissolved in

with an Otsuka DLS-700 dynamic light scatteringchloroform, and the solvent was removed in a rotary

spectrophotometer (Otsuka Electronics, Osaka,evaporator. The lipid mixture was evacuated in a

Japan), and the zeta-potential of the liposomes wasdesiccator under reduced pressure for more than 12

determined with an Otsuka ELS-800 electrophoretich, and an exact amount of lipids was weighed into

light scattering spectrophotometer (Otsuka Elec-the glass vessel of a Potter-homogenizer (a teflon

tronics). Vesicle structure was also investigated byhomogenizer). To this lipid mixture was added a 9%

electron microscopy using a negative staining tech-sucrose aqueous solution (pH 7.0) containing 600 mg

nique [21].of DNA (pbgal-Control) to make a final volume of15 ml and make the final lipid composition ofTMAG/DOPE/DLPC50.4 mM:0.8 mM:0.8 mM (1 3.6. Transfection protocol in vitromg of DNA/10 nmol of TMAG; 40 mg of DNA/mlof the dispersion). After hydration of the lipids by The vials of the lipid-film as the control prepara-


tion method (lipid-film method) in this experiment (Pierce, IL, USA) using bovine serum albumin aswere prepared in a scale of 50 ml as follows: an standard [20].exact amount of chloroform solution of TMAG/DOPE/DLPC in a molar ratio of 2:4:4 was pipettedinto 2 ml glass vials (0.5 ml portion each3100 4. Results and discussionvials), and the solvent was completely removed in adesiccator under reduced pressure for more than 12 h 4.1. Stability of naked DNAafter slight pre-drying in a clean booth. Each vialcontained the lipid composition of TMAG/DOPE/ The influence of pH and temperature on theDLPC5200 nmol:400 nmol:400 nmol, and was stability of circular DNA (pbgal-Control) and linearstored at 2208C until use. For the transfection DNA prepared with Bam III was investigated inexperiment, this lipid-film in the vial was hydrated advance. The following was confirmed in this pre-with 0.5 ml of a 9% sucrose aqueous solution (pH liminary experiment (data not shown): (1) the linear7.0) containing 20 mg of DNA (PGV-C), and the vial DNA was slightly more unstable than the circularwas shaken on a vortex mixer for 10 min at room DNA; (2) the circular DNA was relatively stable intemperature to prepare the liposomes containing the pH range 6.0–10.0 even at 708C, but changeDNA (1 mg of DNA/10 nmol of TMAG; 40 mg of from closed-circular (supercoiled) DNA to open-DNA/ml of the dispersion). It was confirmed from circular DNA was observed after 4 h; (3) DNA wasthe previous experiment that the extrusion procedure very unstable in the FBS-containing medium, thoughwas ineffective due to loss of DNA, so the extrusion it was stable in FBS-free medium; (4) DNA itselfprocedure in order to obtain a more homogeneous was stable, however, even in FBS-containingparticle size distribution was not performed. medium if the liposome/DNA complexes were

R RFor FDEL, an aqueous solution of 20 mg of DNA formed with LipofectAmine or Lipofectin [16].(PGV-C) /465 ml of distilled water was added to the The closed-circular DNA (50 mg of pbgal-Con-FDEL and the vial was shaken by hand at room trol /ml of a 9% sucrose aqueous solution, pH 7.0)temperature in order to obtain the liposomes con- was used for the stability test in this study. Fig. 3taining DNA (1 mg of DNA/10 nmol of TMAG; 40 shows the stability of the naked DNA against variousmg of DNA/ml of the dispersion). procedures used for preparation of liposomes. As

In both cases, the liposomal dispersion was diluted shown in Fig. 3(a), at 658C for 10 min no adhesion40 times with DMEM containing or not containing was observed to vessels such as a polypropylene10% FBS before the transfection experiment, to tube, a round-bottomed glass flask, and a Potter-make a final concentration 1 mg of DNA/ml for the homogenizer. DNA was very unstable under sonica-dispersion. tion (a bath-type sonicator), as shown in Fig. 3(b),

Three kinds of tumor cells (HIRA, HEC-1A and but was relatively stable under vortexing (a vortexColo320DM) were seeded on 6-well plates to about mixer) and homogenization (a Potter-homogenizer),

5 630–50% confluence (10 –10 cells in a well). Cells with only minor conformational change, if any,were washed with serum-free DMEM, and 1 ml of observed. It thus appears that closed-circular DNAthe above liposomal dispersion (1 mg of DNA/ml of surrounded by a water shell can avoid the shearthe dispersion) was added to start the transfection. stress of emulsification with vortex mixers and teflonAfter incubation at 378C for 5 h, cells were washed homogenizers. Next, the influence of filtrationand DMEM containing 10% FBS was added. After through membrane filter was examined. As shown incultivation for 2 days, luciferase activities were Fig. 3(c), no degradation or loss of naked DNA wasassayed using the Pica gene luciferase assay kit observed after filtration through the PVDF mem-(Toyo Ink, Tokyo, Japan). Light emission was brane filter (pore size, 0.45 and 0.22 mm), though ameasured with a lumiphotometer, and luciferase slight conformational change was detected with useactivity was expressed as light units per second per 1 of the CE membrane filter (pore size, 0.22 mm). Itmg protein of cell extracts. Protein concentrations thus appears that the internal structure of the mem-were determined by BCA Protein Assay Reagents brane filters is important, and that closed-circular


Fig. 3. Stability of naked DNA (pbgal-Control, 50 mg/ml) against various procedures: (a) adhesion of DNA to a polypropylene tube, around-bottomed glass flask or a Potter-homogenizer (teflon pestle and glass vessel) at 658C for 10 min; (b) influence of emulsification by abath-type sonicator, a vortex mixer or a teflon homogenizer at 658C for 10 min; (c) influence of filtration through a PVDF membrane filter(pore size, 0.45 or 0.22 mm) or a CE membrane filter (pore size, 0.22 mm) used repeatedly in triplicate. After the above treatment, the nakedDNA solution was diluted ten times and 10 ml of the diluted DNA solution (50 ng of DNA as the initial amount) was applied to gelelectrophoresis. An Intelligent Quantifier was used for determination of the quantity of closed-circular (supercoiled) DNA and open-circularDNA. The vertical axes in each figure shows the residual closed-circular DNA and open-circular DNA.

DNA undergoes the shear stress when it penetrates brane filter, due to strong complex formation withthrough the CE membrane filter. the cationic liposomes.

With the FDEL method, on the other hand, no4.2. Stability of DNA during preparation of degradation, conformational change or loss of DNAliposomes was observed, since the homogenization and extru-

sion procedure were not used after the addition ofIt was confirmed from the above experiments that DNA aqueous solution.

naked DNA itself was relatively stable against When pCAG-LacZ was used instead of pbgal-various procedures used for preparation of lipo- Control, nearly the same results were obtained (datasomes, but the following experiments showed that not shown).DNA was not stable when liposomes were actually The above findings reveal a large advantage of theprepared by the conventional method. FDEL method, since for mass production of lipo-

Fig. 4 shows the stability of DNA (pbgal-Control) somes containing DNA which will be used for induring preparation of the liposomes composed of vivo gene therapy, procedures for homogenizationTMAG/DOPE/DLPC50.4 mM:0.8 mM:0.8 mM. and extrusion are needed to obtain a homogeneousWhen the liposomes containing DNA were prepared particle size distribution. With the conventional lipid-by the lipid-film method, significant degradation and film method, significant degradation and conforma-conformational change of DNA was observed during tional change of DNA were observed during thehomogenization. The loss of DNA was also signifi- procedures for sizing. With the FDEL method, on thecant after the first extrusion for sizing and steriliza- other hand, the procedures for homogenization andtion, and residual DNA in the final preparation after extrusion have been already performed prior tothe second extrusion was barely detected; in fact, this addition of a DNA aqueous solution, and thus thefinal preparation had no transfection activity. These stability of DNA is not a concern.findings were considered due to the strong inter-action between the cationic liposomes and the 4.3. Characterization of the liposomes prepared bynegatively-charged DNA. It thus appeared that DNA the FDEL methodwas affected by the shear stress of the teflonhomogenizer and the internal structure of the mem- The newly-developed FDEL method was applied


to many types of cationic liposomes and plasmidDNAs, and it was confirmed by characterization ofthese liposomes that this method has additionaladvantages over the conventional lipid- film method:(1) the liposomes can be sterilized, since filtration(pore size, 0.22 mm) of the empty liposomal disper-sion is very easy (data not shown); (2) the stability ofFDEL is excellent during storage, because the lipo-somes are freeze-dried (data not shown); (3) repro-ducibility of the results of experiment is very good,since a large number of vials of the same lot can besupplied (data not shown); (4) the particle size iswell controlled in advance, as shown in Fig. 5. Theparticle size distribution of the liposomes as de-termined by dynamic light scattering spectrophotom-etry was 100 to 200 nm for almost all of theliposomal formulations tested (data not shown), andthe presence of DNA and FBS did not practicallyaffect the particle size (data not shown).

Fig. 4. Stability of DNA (pbgal-Control) during preparation of 4.4. Transfection activity of the liposomesliposomes by the lipid-film method and the FDEL method. The

prepared by the FDEL methodlipid composition was TMAG/DOPE/DLPC50.4 mM:0.8mM:0.8 mM, and the preparation scales were 15 ml for thelipid-film method and 500 ml (0.5 ml31000 vials) for the FDEL Fig. 6 shows luciferase activities after transfectionmethod. After extraction of DNA from the liposomal dispersion, a of three kinds of tumor cells, HRA, HEC-1A anddefinite amount of extracted DNA solution (50 ng of DNA as the Colo320DM, by liposomes containing DNA (PGV-initial amount) was applied to gel electrophoresis: M, molecular

C).weight ladder (lHindIII); I, initial; H, after homogenization; E1,For the liposomes prepared by the lipid-filmafter first extrusion; E2, after second extrusion; R, after rehydra-

tion of the FDEL by the DNA solution and shaking. method, vortexing was vigorously performed but the

Fig. 5. Negative-stain electron micrographs of the liposomes prepared by (a) the lipid-film method, and (b) the FDEL method. The lipidcomposition was TMAG/DOPE/DLPC50.4 mM:0.8 mM:0.8 mM, and both lipid-film and FDEL were rehydrated with distilled water.


and efficiency even in the presence of FBS thancommercialized reagents for transfecticin [20].

5. Conclusions

Our experimental findings suggest that the freeze-dried empty liposomes (FDEL) method is very usefulfor preparation of liposomes containing DNA andliposome/DNA complexes, which can be used notonly for in vitro gene transfer but also for in vivo

Fig. 6. Luciferase activity after transfection of three kinds ofgene therapy, since DNA is very stable and thetumor cells by two kind of liposomes containing DNA (PGV-C),particle size of the liposomes can be controlled wellwhich were prepared by the lipid-film method and the FDEL

method: The lipid composition was TMAG/DOPE/DLPC50.4 during the preparation procedure.mM:0.8 mM:0.8 mM, and 1 mg of DNA per 10 nmol of TMAG

5 6was added to 10 –10 tumor cells in medium with /without 10%of FBS.

Acknowledgements

extrusion procedure was not performed, since it The authors would like to thank all collaborators,would have caused loss of DNA. The luciferase especially Dr. Masahiro Nishijima and Dr. Takayukiactivity in the presence of 10% FBS was much Kitagawa of the National Institute of Health (Japan),higher than that in the absence of FBS in all tumor and also Dr. Hideaki Tahara, University of Pitts-cells tested: it is well known that the lipid com- burgh Medical Center, for many helpful discussionsposition of TMAG/DOPE/DLPC in a molar ratio of and much advice.2:4:4 has great transfection activity even in thepresence of FBS [19].

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Gene delivery using liposome technology