Lyophilization of cationic lipid–protamine–DNA (LPD) complexes

Lyophilization of Cationic Lipid–Protamine–DNA(LPD) Complexes

BEI LI,1* SONG LI,2 YADI TAN,2 DONNA B. STOLZ,3 SIMON C. WATKINS,3 LAWRENCE H. BLOCK,1 LEAF HUANG2

1 Graduate School of Pharmaceutical Sciences, School of Pharmacy, Duquesne University,Pittsburgh, Pennsylvania 15282

2 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

3 Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh,Pittsburgh, Pennsylvania 15261

Received 18 August 1999; accepted 1 December 1999

ABSTRACT: Cationic lipid-based gene delivery systems have shown promise in trans-fecting cells both in vitro and in vivo. However, these systems tend to form aggregatesin liquid formulation during storage, which has limited their clinical applications. As aresult, lyophilization of these systems has recently become a subject of increasinginterest. In this paper, lyophilization of LPD, a novel cationic lipid-based gene deliverysystem, was studied. Both particle size and transfection efficiency could be preserved inthe presence of sufficient amount of appropriate lyoprotectant. A series of monosaccha-rides and disaccharides, including dextrose, galactose, mannose, lactose, maltose, su-crose and trehalose, were evaluated for their lyoprotective effect and disaccharidesshowed more superior protection to monosaccharides. The effect of different freezingprotocols for lyophilization was also evaluated and no significant difference was found.However, for freeze-thawing, fast freezing caused less aggregation. Finally, nonlyophi-lized LPD and LPD lyophilized with 10% sucrose were stored at different temperaturesand their stability was followed for eight weeks. Lyophilized LPD could be stored atroom temperature without significant change in particle size or loss of transfectionefficiency. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89:355–364, 2000Keywords: gene therapy; nonviral vector; lipopolyplex; lyophilization

INTRODUCTION

Gene therapy represents an important advance inthe treatment of both inherited and acquired dis-eases.1,2 However, success of human gene therapy

largely depends upon the development of deliveryvehicles or vectors which can selectively delivertherapeutic genes to target cells with efficiencyand safety. Viral vectors, although highly effi-cient, suffer from a number of problems such asimmunogenicity,3 toxicity,4 and potential recom-bination or complementation.5 As a result of theselimitations, much effort has been devoted to thedevelopment of nonviral vectors, such as cationicliposomes6,7 and cationic polymers.8,9 Cationic li-posomes are particularly attractive due to theirfavorable characteristics such as biocompatibil-ity, minimal toxicity, lack of specific immune re-

Correspondence to: L. Huang, Department of Pharmaceu-tical Sciences, University of Pittsburgh School of Pharmacy,W1351 Biomedical Science Tower, Pittsburgh, PA 15261.(E-mail: [email protected])

*Present address: Department of Pharmaceutical Chemis-try, The University of Kansas, 2095 Constant Avenue,Lawrence, KS 66047Journal of Pharmaceutical Sciences, Vol. 89, 355–364 (2000)© 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 3, MARCH 2000 355

sponse, relative ease of large-scale production,and simplicity of use. Since the description of thefirst cationic lipid by Felgner in 1987,10 numerousnew lipids have been reported.6,7 Currently, cat-ionic liposomes are widely employed for the trans-fection of eukaryotic cells in research laborato-ries. Several liposomal formulations have also un-dergone clinical evaluation as vectors for genetherapy in cancer and cystic fibrosis.11–13

Despite the potential of cationic lipid vectors ingene therapy, their efficiency needs to be im-proved to achieve clinical benefit. Also, the stabil-ity of these systems needs to be improved beforecationic lipid-mediated gene therapy can becomea routine practice in the clinic.14 Most liposomalformulations that have been developed in genedelivery are essentially colloidal systems, andthermodynamic instability is an inherent charac-teristic of these kinds of system. Cationic lipid/DNA complexes tend to form aggregates, result-ing in a decrease in transfection efficiency.15,16

Because of this problem, cationic lipid/DNA com-plexes have to be freshly prepared at bedsideprior to being applied to patients.12,17 This willnot only require training of physicians but alsomake quality control very difficult due to the factthat preparation of cationic lipid/DNA complexesis a process that is poorly defined and difficult tocontrol.

To develop stable gene delivery systems thatare suitable for long-term storage, lyophilizationappears to be a feasible approach. Several studieshave demonstrated that gene delivery systemscan be lyophilized without affecting their physicalcharacteristics and transfection efficiency invitro.18–20 In this study, lyophilization of cationiclipidic vectors is further investigated using LPDas a model. LPD is a novel lipidic vector that wasrecently developed in this laboratory. These nano-particles, about 100 nm in diameter, contain acore of protamine-condensed DNA coated with alipidic shell, very much like enveloped viruses.Compared with cationic lipid/DNA complexes,LPD is much more stable and also more efficientin transfecting cells in vitro.21 Recently, LPD hasalso been shown to be highly efficient in genetransfer in vivo upon intracranial injection or sys-temic administration.22,23 The goals of this studyare to determine the feasibility of preserving thephysical stability (particle size) and transfectionefficiency of LPD through lyophilization and toidentify the factors that affect the stability ofLPD. Several carbohydrates were examined fortheir lyoprotectant activity during lyophilization

of LPD. We also examined the effect of differentfreezing protocols on lyophilization. Finally, thestability of lyophilized and nonlyophilized LPD atdifferent temperatures was followed over 8 weeksin terms of particle size of LPD dispersion and itstransfection efficiency.

MATERIALS AND METHODS

Chemicals

1,2-Dioleoyl-3-trimethylammoniumpropane(DOTAP) was purchased from Avanti Polar Lip-ids (Alabaster, AL). Cholesterol, sucrose, treha-lose, maltose, lactose, dextrose, galactose, andmannose were obtained from Sigma (St. Louis,MO). Protamine sulfate USP was from Eli Lilly(Indianapolis, IN). Luciferase assay kit was ob-tained from Promega (Madison, WI), and Coo-massie Plus protein assay reagent was purchasedfrom Pierce (Rockford, IL). All other chemicalswere of reagent grade.

Purification and Iodination of Plasmids

Plasmid pCMVL, which contains the cDNA offirefly luciferase driven by a human cytomegalo-virus immediate-early promoter, was amplified inDH5a strain of Escherichia coli, isolated and pu-rified using an EndoFree Plasmid Giga kit fromQIAGEN (Valencia, CA). Plasmid DNA was la-beled with 125I by using a published method24 andpurified by a spin column (Bio-Spin-P30; Bio-Rad,Hercules, CA).

Preparation of Liposomes

Liposomes containing DOTAP and cholesterol ina 1:1 molar ratio were prepared as described by Liet al.23 The lipid mixture in chloroform was driedto a thin film with a stream of N2 gas and wasfurther desiccated under vacuum for 2 h. Thelipid film was hydrated in water to give a finalconcentration of 10 mg DOTAP/mL. The lipid sus-pension was incubated at 50°C for 10 min andthen sequentially extruded through polycarbon-ate membranes (Corning Separations Division,Acton, MA) with pore sizes of 0.6 and 0.2 mm.

Preparation and Lyophilization of LPD

LPD was prepared according to a protocol de-scribed by Li et al.23 Briefly, diluted DNA wasadded to the mixture of DOTAP/cholesterol lipo-

356 LI ET AL.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 3, MARCH 2000

somes and protamine at a final ratio of 8.6 mgDOTAP/0.6 mg protamin/mg DNA. The mixturewas incubated at room temperature for 20 min forthe formation of complexes. For the experimentwhere free liposomes needed to be removed fromthe dispersion, 125I-labeled DNA was preparedand used for the preparation of LPD. The disper-sion was subjected to sucrose gradient ultracen-trifugation and DNA enriched fraction was col-lected as described previously.23 Sucrose wasthen removed by ultrafiltration, and the DNAconcentration was adjusted to be the same as theoriginal dispersion based on radioactivity. Beforelyophilization, the dispersion was diluted (1:1,v/v) with water or with aqueous solution of thedifferent sugars, and aliquots of 300–600 ml wereadded to Wheaton serum tubing vials (5 mL).They were frozen using one of the following pro-tocols: at −20°C for 30 min and then at −80°C for30 min; at −80°C for 60 min; in liquid nitrogen for2 min. The various samples were then lyophilizedfor 24 h on a Labconco freeze-dryer (Model 4.5).After lyophilization, vials were sealed with rub-ber stoppers and aluminum seals. Prior to themeasurement of particle size and transfection ef-ficiency, the lyophilized samples were resus-pended with distilled water to the original vol-ume. DNA recovery was followed by 125I-labeledDNA.

Particle Size Measurement

The particle size of LPD was determined by dy-namic laser light scattering using a Coulter N4SDparticle sizer (Hialeah, FL). Three successive sizedeterminations were made on each sample.

Negative Stain Electron Microscopy

Copper grids (200 mesh) were Formvar coated us-ing 0.125% Formvar in chloroform. Grids arefloated on a drop of undiluted LPD sample forapproximately 30 s. The grid is removed, and ex-cess sample solution is wicked away with filterpaper. Grid is then placed on a drop of 0.45-mmfiltered 4% uranyl acetate in Milli-Q H2O for 30–60 s. Excess stain is wicked away, and samplesare viewed on a JEOL JEM 1210 transmissionelectron microscope at 80 kV.

Cell Culture and In Vitro Transfection

BL-6 cells were cultured in RPMI 1640 mediumcontaining 10% fetal bovine serum supplemented

with antibiotics. Cells grown in a 48-well plate ofabout 80% confluency were washed with serum-free medium once and then incubated for 4 h witha dilution of the LPD dispersion before the me-dium was replaced with RPMI 1640 containingserum. LPD was diluted with serum-free mediumto 1 mg DNA/mL and 0.5 mL of this dilution wasadded to each well. Thirty-six hours later, cellswere washed with saline and lysed for 5 min atroom temperature with 200 mL lysis buffer (0.05%Triton X-100, 2 mM EDTA, 0.1 M Tris, pH 7.8).Cell lysates were then centrifuged at 14,000g for5 min at 4°C. A 20 mL of the supernatant wasmeasured for protein concentration with Coo-massie Plus protein assay reagent according tothe manufacturer’s protocol and another 20 mLwas analyzed for luciferase activity using an Au-tomated LB 953 luminometer equipped with anautomated injector (Berthold, Bad Wildbad, Ger-many). The results were expressed as relativelight units (RLU) per mg protein.

In vivo Transfection

Female CD1 mice of 4–6 weeks of age were pur-chased from Charles River Laboratories (Wil-mington, MA) and were housed in accordancewith institutional guidelines. Individual mice ingroups of three were injected via tail vein with 25mg of DNA formulated in LPD in a total volume of300 mL. Twenty-four hours following i.v. injection,the mice were killed by cervical dislocation andheart, lung, spleen, liver, and kidney were col-lected. The organs were washed with cold salinetwice and homogenized with lysis buffer using atissue tearor (Biospec Products, Bartlesville, OK).The homogenates were centrifuged at 14,000g for10 min at 4°C. The supernatant was analyzed forluciferase activity and protein content as de-scribed above.

RESULTS

Sucrose, a frequently used lyoprotectant, wasfirst tested for its use in the lyophilization of LPD.Several aliquots from the same batch of LPD werediluted with water or concentrated sucrose solu-tion to a final sucrose concentration of 1%, 2.5%,5%, or 10%. The dilutions were then frozen inliquid nitrogen for 2 min and lyophilized over-night. After lyophilization, samples with sucroseof 5% and 10% formed cakes that could be easilyredispersed in water. Particle sizes of the samples

LYOPHILIZATION OF LPD 357


were measured by dynamic laser light scatteringand in vitro transfection efficiency was tested inBL-6 cells. As shown in Figure 1, lyophilization ofLPD without sucrose or with 1% sucrose resultedin a significant increase in size. The size of recon-stituted LPD became well preserved when theconcentration of sucrose increased to 2.5% orgreater. The change in size was confirmed bynegative-stain EM (Figure 2). Lyophilization with1% of sucrose or without sucrose led to aggrega-tion of LPD, which involved most, if not all, of theparticles. LPD lyophilized with 5% of sucroseshowed structures similar to those of nonlyophi-lized LPD. Transfection efficiency of LPD in-versely correlated with particle size. Increase insize was associated with a decrease in transfec-tion efficiency. When the particle size was pre-served, the transfection efficiency was also re-tained. Lyophilization did not significantly affectthe configuration of plasmid DNA when LPD sizewas well preserved (data not shown).

The lyoprotective effect of several other carbo-hydrates was also investigated for three differentfreezing protocols. LPD was prepared and dilutedwith a 20% sugar solution (1:1, v/v) to a final con-centration of 10%, the level at which sucrose hadshown an optimal effect. Each sample was further

divided into four aliquots. One of the aliquots waskept at 4°C while the other three were frozen us-ing different protocols before lyophilization: at−20°C for 30 min and then at −80°C for 30 min; at−80°C for 60 min; in liquid nitrogen for 2 min.Figure 3 shows the effect of three monosacchari-des: dextrose, galactose, and mannose. There wasno marked difference among the three differentfreezing protocols with respect to preservation ofboth particle size and transfection efficiency. Thetransfection efficiency was retained regardless ofwhich sugar or freezing condition was used. How-ever, an increase in particle size was found in allsamples although the size increases were smallerthan in the absence of the sugars.

Several disaccharides including lactose, malt-ose, sucrose and trehalose were also tested fortheir lyoprotective effect (Figure 4). The one-stepfreezing protocol (−80°C for 60 min) was used be-fore lyophilization. Again, transfection efficiencywas retained in all samples. However, comparedwith monosaccharides, particle size was betterpreserved when disaccharides were used. Theonly exception was lactose, which failed to fullypreserve the particle size when the slow freezingmethod was used. Although maltose proved excel-lent in preserving both particle size and transfec-tion efficiency, it is a reducing sugar and mightenter into secondary browning reactions thatcould destabilize the formulation. Therefore, sub-sequent studies were focused on sucrose and tre-halose.

Figure 5 shows in vivo gene expression of LPDbefore and after lyophilization with 10% sucroseor trehalose. The one-step freezing protocol(−80°C for 60 min) was used before lyophilization.Twenty-five mg of DNA was injected intrave-nously into each mouse, and luciferase activity inheart, lung, liver, spleen, and kidney was exam-ined 24 h later. Lyophilized and nonlyophilizedsamples were similar with respect to the level andpattern of gene expression: gene expression wasdetected in all major organs including heart, lung,liver, spleen, and kidney, with the lung havingthe highest level of gene expression.

For each of the different freezing protocols, weinvestigated the effect of freeze–thawing on LPDparticle size in the presence of various concentra-tions of the cryoprotectants: sucrose and treha-lose. Samples frozen by each of the three differentfreezing protocols described above were thawed at4°C in a refrigerator overnight. When the data inFigure 6 are contrasted with the data in Figure 1,it is evident that freeze–thawing is a less stressful

Figure 1. Effect of lyophilization on in vitro transfec-tion efficiency and on particle size of LPD as a functionof sucrose concentration. In vitro transfection efficiencywas expressed as the percentage of that of freshly madeLPD. Each data point represents the mean ± SD ofthree measurements. The experiment was repeated 2times with essentially the same results.

358 LI ET AL.


process than freeze-drying. When the sample wasfrozen in liquid nitrogen, particle size stayed al-most unchanged even without any cryoprotec-tant. Aggregation was observed when thesamples were frozen using the other two proto-cols. However, particle size was fully preservedwhen as little as 1% sucrose was used. Similarresults were observed when trehalose was used.

We have previously shown that LPD is hetero-geneous in population, containing free liposomesand small amounts of lipid/DNA complexes in ad-dition to lipid–protamine–DNA complexes.23 Thenecessity of free liposomes for in vivo transfectionvaries according to different administrationroutes.25 While free liposomes are essential forLPD to achieve maximal gene expression via in-travenous administration, gene transfer via in-tratissue injection will require removal of free li-posomes. To investigate the effect of lyophiliza-tion on the stability of LPD in the absence of freeliposomes, free liposomes were removed fromLPD by sucrose gradient centrifugation. The ef-

fect of lyophilization on the particle size of puri-fied LPD was then evaluated as described above.Liposome/DNA complexes and liposome alonewere used as controls. As shown in Figure 7, thepurified complexes exhibited a greater stabilitythan the unpurified ones (compared with Figure1). As little as 1% sucrose is sufficient to fullypreserve the particle size of purified LPD. Therewas no significant difference between the purifiedLPD and liposome/DNA complex. Furthermore,cationic liposome alone exhibited a similar sensi-tivity to sucrose as a lyoprotectant as compared tothe purified LPD and liposome/DNA complex, ex-cept the fact that the size of the former was some-what smaller than that of the latter ones.

Figure 8 shows the stability of LPD stored atdifferent temperature for prolonged periods oftime. A 200-mL amount of LPD in 10% sucrosewas aliquoted into 3-mL glass vials. The vialswere sealed with a rubber stopper and aluminumseal either directly or after lyophilization. Theywere kept at 4°C, room temperature, or 37°C for 8

Figure 2. Electron micrographs of LPD before and after lyophilization. LPD, nonly-ophilized (A), lyophilized with 0% (B), 1% (C), and 5% (D) were subjected to electronmicroscopic examination as described in Materials and Methods. (A, D) Samples dis-play both empty liposomes (arrow) and condensed particles (arrowheads) that are notclumped. (B, C) Particles are clumped into larger aggregates (arrows).



weeks. Particle size and in vitro transfection effi-ciency were measured at different time intervals.Transfection efficiency was expressed as percent-age of that of freshly prepared LPD. The nonly-ophilized samples kept at 37°C formed large ag-gregates (∼3,000 nm) by the end of the first week(data not shown), and their transfection efficiencydropped to less than 1%. Thus, they were not fol-lowed further. For the nonlyophilized samplesstored at room temperature, there was a gradualincrease in the size of LPD over the first 4 weeksand large aggregates (∼3,000 nm) began to appearthereafter. Transfection efficiency decreased toapproximately 50% over the first 2 weeks andthen dropped to less than 1% after the fourthweek. For the nonlyophilized samples stored at4°C, particle size was well retained over the 8weeks with little change in transfection efficiencyover the same period of time. The size and trans-

fection efficiency of lyophilized LPD was well re-tained over the 8 weeks. Only a slight decrease intransfection was noticed in lyophilized LPD thatwas stored at room temperature.

In this study, the residual moisture content inthe lyophilized samples was also measured usingthe Karl Fisher method. The moisture contentranged from 1% to 5% (w/w) in the samples lyoph-ilized with sucrose and from 0.5% to 4% (w/w) inLPD lyophilized with trehalose. However, no cor-relation was found between moisture content andparticle size or transfection efficiency (data notshown). There were no significant changes in themoisture content of lyophilized LPD over an8-week period of time. The physical appearancealso remained unchanged over the same period oftime (data not shown).

DISCUSSION

Lyophilization is frequently used to stabilize la-bile pharmaceutical agents such as protein and

Figure 3. Effect of monosaccharides and freeze pro-tocols on particle size (A) and in vitro transfection effi-ciency (B) of lyophilized LPD Three freeze protocolswere employed for lyophilization: at −20°C for 30 minand then at −80°C for 30 min (dotted bars), at −80°C for60 min (blank bars) and in liquid nitrogen for 2 min(hatched bars). The black bars represent samples with-out lyophilization. The concentration of all monosac-charides was 10%. Each bar represents the mean ± SDof three measurements. The experiment was repeated 2times with essentially the same results.

Figure 4. Effect of disaccharides and freeze methodson particle size (A) and in vitro transfection efficiency(B) of lyophilized LPD. Three freeze protocols were em-ployed for lyophilization: at −20°C for 30 min and thenat −80°C for 30 min (dotted bars), in −80°C for 60 min(blank bars) and in liquid nitrogen for 2 min (hatchedbars). The black bars represent samples without ly-ophilization. The concentration of all disaccharides was10%. Each bar represents the mean ± SD of three mea-surements. The experiment was repeated 3 times withessentially the same results.

360 LI ET AL.


drug-loaded liposomes. However, the lyophiliza-tion process itself can damage biomolecules un-less appropriate lyoprotectants are utilized. Di-saccharides, such as sucrose and trehalose, have

been extensively studied for their ability to pre-serve size and membrane permeability of lipo-somes during lyophilization.26,27 It has been pro-posed that during freezing, sugars have the abil-ity to form a rigid sugar-glass or vitrified networkstructure in which liposomes can be embedded.This vitrified network preserves of the integrity ofthe liposomes.26,27 Sugars have also been shownto interact with lipid bilayer directly through H-bonding in dry state, which provide a steric bar-rier to bilayer apposition.26,27 Similar interactionmay also take place during freezing. This wassupported by the study of Anchordoguy et al.,28

which suggested an interaction of lyoprotectantswith the acyl chains of liposome bilayer duringfreezing. Another important effect of sugars isthat they can significantly decrease the phasetransition temperature of lipids at dry state,which prevents phase transition during lyophili-zation and leakage during rehydration.26,27 How-ever, previous studies have mainly focused on ly-ophilization of anionic or neutral liposomes. Littleis known about the stability of cationic liposomesand cationic liposome/DNA complexes subjectedto lyophilization.

Like anionic or neutral liposomes, cationic li-posomes are spherical vesicles. However, there isa substantial change in the structure of liposomesafter their interaction with plasmid DNA. The re-

Figure 5. In vivo gene expression following i.v. ad-ministration of LPD containing 25 mg pCMVL DNA.Each bar represents the mean ± SD for three mice.

Figure 6. Effect of freeze–thawing on particle size ofLPD. LPD was frozen according to different protocolsand thawed at 4°C in the absence and presence of vari-ous concentrations of sucrose. Three freeze protocolswere employed: at −20°C for 30 min and then at −80°Cfor 30 min (s), at −80°C for 60 min (h) and in liquidnitrogen for 2 min (n). The filled circle represents theunfrozen sample. Each data point represents the mean± SD of three measurements. The experiment was re-peated 2 times with essentially the same results.

Figure 7. Effect of lyophilization on particle size ofpurified LPD, DOTAP–cholesterol liposome and DOT-AP–cholesterol liposome/DNA complex. Each datapoint represents the mean ± SD of three measure-ments.



sulting liposome/DNA complexes bear little re-semblance to the starting liposomes. Dependingon the structure of the cationic lipid, the lipid/DNA complexes may appear as large sphericalaggregates, tubular structures or small con-densed particles.29 Thus, maintenance of the per-meability of the lipid bilayer is not a major con-cern in the lyophilization of cationic lipid/DNAcomplexes. Rather, preservation of the particlesize is critical as the transfection efficiency of li-pidic vectors is greatly affected by their size.30,31

As shown in Figure 1, maintenance of the size ofLPD was associated with a full retention of trans-fection efficiency. This is in agreement with a pre-vious study by Anchordoquy et al. for lipid/DNAcomplexes.18

The role of saccharides in lyophilization of LPDis not clear. It may involve a mechanism similarto that in lyophilization of anionic or neutral li-posomes, i.e., vitrification and formation of asteric barrier via H-bonding with the lipid bi-layer. This may explain why monosacchrideswere inferior to disaccharides with respect topreservation of particle size. A previous studysuggested that monosaccharides were less effec-tive than disaccharide in forming H-bonding withlipids.32 In addition, due to their relativelysmaller molecular size as compared to disaccha-

rides, monosaccharide-mediated formation ofsteric barriers may be less effective.

The effect of freeze-thawing on the size of cat-ionic lipid/DNA complexes has been studied byAnchordoquy et al.33 It was found that the size ofcomplexes was better preserved when the com-plexes were frozen rapidly rather than slowly. Itwas explained that aggregation was promoted byconcentration of the components during slowfreezing. Similar results were found in this studywith LPD (Figure 6). Interestingly, much lowerconcentrations of saccharides were required topreserve the size of LPD in freeze–thawing ascompared with lyophilization. These results sug-gest that while saccharides play an importantrole in maintaining the size in the initial freezingstep, they play more important roles in the sub-sequent hydration and rehydration steps.

The LPD examined in this study was optimizedfor in vivo gene transfer via intravenous admin-istration. We have previously shown that LPD isa heterogeneous population that contains excessamounts of free liposomes and also smallamounts of lipid/DNA complexes.23 While the freeliposomes are essential for maximal gene expres-sion in intravenous gene delivery, they haveproven deleterious when other administrationroutes such as intracranial injection are chosen.25

Figure 8. Stability of LPD with 10% sucrose stored at different temperature in termsof particle size (A) and in vitro transfection efficiency on BL-6 cell line (B). Each datapoint presents the mean ± SD of three measurements.

362 LI ET AL.


Under these circumstances, free liposomes needto be removed by sucrose-gradient centrifugation.Results in Figure 7 demonstrate that purifiedLPD can also be lyophilized without adversely af-fecting its size. More interestingly, comparedwith unpurified LPD, lower concentrations of sac-charides were required for lyophilization of puri-fied LPD. The stability of purified LPD is similarto that of cationic liposome alone (Figure 7). Theimproved stability of purified LPD might de dueto, among others, their unique structure. Unlikecationic lipid/DNA complexes that form varioustypes of aggregates differing in sizes, LPD appearas small, compact particles as shown by negativestaining- and cryo-EM. They contain a core ofprotamine-condensed DNA coated with lipid bi-layers (Li et al., manuscript in preparation).These highly compact and lipid-enveloped struc-tures may play an important role in improvingthe colloidal stability of LPD.

The stability of nonlyophilized LPD and lyoph-ilized LPD during storage was also studied. Non-lyophilized LPD was highly sensitive to storagetemperature. This was not surprising consideringthe colloidal nature of the formulation. However,when stored at 4°C, the LPD dispersion was rela-tively stable: over a period of 8 weeks, no aggre-gates were noticed and transfection efficiency wasmaintained. This is quite different from lipid/DNA complexes without protamine, which readilyform aggregates during storage.33 Despite therelatively stable nature of LPD at 4°C, lyophili-zation is still advantageous for the following rea-sons: (1) transfection efficiency of lyophilizedsamples was higher than nonlyophilized samples;and (2) lyophilized samples were less sensitive totemperature. Even when the samples were storedat room temperature, there was no significantloss in transfection efficiency over a period ofeight weeks. This opens the possibility of storingand shipping the formulation at room tempera-ture. Finally, in the lyophilized state in contrastto the liquid state, the mobility and reactivity ofthe components is dramatically reduced. Thismay increase the chemical stability of the formu-lation although it is not addressed in the currentstudy.

In conclusion, our study shows that preserva-tion of particle size is important in maintainingtransfection efficiency during lyophilization. Di-saccharides were superior to monosaccharides inpreventing aggregation. Lyophilization wasproven to be suitable for preserving both the par-ticle size and transfection efficiency of LPD in the

presence of an appropriate lyoprotectant. LPD ly-ophilized with 10% sucrose could be stored atroom temperature without marked change in par-ticle size or loss of transfection efficiency. Furtherlong-term stability studies are required to deter-mine the shelf life of lyophilized LPD at roomtemperature.

ABBREVIATIONS

LPD cationic lipid-protamine-DNA com-plexes

DOTAP 1,2-dioleoyl-3-trimethylammoniumpro-pane

ACKNOWLEDGMENTS

This work was supported by NIH GrantsDK54225 and CA 74918 and a grant from the Tar-geted Genetics Corporation to L.H.

REFERENCES AND NOTES

1. Miller AD. 1992. Human gene therapy comes ofage. Nature 357:455–460.

2. Ross G, Erickson R, Knorr D, Motulsky AG, Park-man R, Samulski J, Straus SE, Smith BR. 1996.Gene therapy in the United States: a 5 year statusreport. Hum Gene Ther 7:1781–1790.

3. Herz J, Gerard RD. 1993. Adenovirus mediatedtransfer of low density lipoprotein receptor geneacutely accelerates cholesterol clearance in normalmice. Proc Natl Acad Sci USA 90:2812–2816.

4. Simon RH, Engelhardt JF, Yang Y, Zepeda M, We-ber-Pendleton S, Grossman M, Wilson JM. 1993.Adenovirus mediated transfer of the CFTR gene tolung of nonhuman primates: toxicity study. HumGene Ther 4:771–780.

5. Ali M, Lemoine NR, Ring CJA. 1994. The use ofDNA virus as vectors for gene therapy. Gene Ther1:367–384.

6. Gao X, Huang L. 1995. Cationic liposome mediatedgene transfer. Gene Ther 2:710–722.

7. Felgner PL. 1996. Improvements in cationic lipo-somes for in vivo gene transfer. Hum Gene Ther7:1791–1793.

8. Wu GY, Wu CH. 1988. Receptor mediated gene de-livery and expression in vivo. J Biol Chem 263:14621–14624.

9. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD,Scherman D, Demeneix B, Behr JP. 1995. A versa-tile vector for gene and oligonucleotide transferinto cells in culture and in vivo: polyethylenimine.Proc Natl Acad Sci USA 92:7297–7301.



10. Felgner PL, Gadek TR, Holm M, Roman R, ChanHW, Wenz M, Northrop JP, Ringold GM,Danielsen M. 1987. Lipofection: a highly efficient,lipid mediated DNA transfection procedure. ProcNatl Acad Sci USA 84:7413–7417.

11. Nabel GJ, Nabel EG, Yang ZY, Fox BA, Plautz GE,Gao X, Huang L, Shu S, Gordon D, Chang AE.1993. Direct gene transfer with DNA liposomecomplexes in melanoma: expression, biological ac-tivity, and lack of toxicity in humans. Proc NatlAcad Sci USA 90:11307–11311.

12. Caplen NJ, Alton EWFW, Middleton PG, Dorin JR,Stevenson BJ, Gao X, Durham SR, Jeffrey PK,Hodson ME, Coutelle C, Huang L, Porteous DJ,Williamson R, Geddes DM. 1995. Liposome medi-ated CFTR gene transfer to the nasal epithelium ofpatients with cystic fibrosis. Nat Med 1:39–46.

13. McLachlan G, Ho L-P, Davidson-Smith H, Sam-ways J, Davidson H, Stevenson BJ, Carothers AD,Alton EWFW, Middleton PG, Smith SN, KallmeyerG, Michaelis U, Seeber S, Naujoks K, Greening AP,Innes JA, Dorin JR, Porteous DJ. 1996. Laboratoryand clinical studies in support of cystic fibrosisgene therapy using pCMVCFTRDOTAP. GeneTher 3:1113–1123.

14. Middaugh CR, Evans RK, Montgomery DL, Ca-simiro DR. 1998. Analysis of plasmid DNA from apharmaceutical perspective. J Pharm Sci 87:130–146.

15. Felgner PL, Tsai YJ, Sukhu L, Wheeler CJ, Man-thorpe M, Marshall J, Cheng SH. 1995. Improvedcationic lipid formulations for in vivo gene therapy.Ann NY Acad Sci 1995;772:126–139.

16. Gustafsson J, Arvidson G, Karlsson G, Almgren M.1995. Complexes between cationic liposomes andDNA visualized by Cryo-TEM. Biochim BiophysActa 1235:305–312.

17. Nabel GJ, Chang A, Nabel EG, Plautz G, Fox BA,Huang L, Shu S. 1997. Immunotherapy of malig-nancy by in vivo gene transfer into tumors. HumGene Ther 3:399–410.

18. Anchordoquy TJ, Carpenter JF, Kroll DJ. 1997.Maintenance of transfection rates and physicalcharacterization of Lipid/DNA complexes afterfreeze-drying and rehydration. Arch Biochem Bio-phys 348:199–206.

19. Cherng JY, Van de Wetering P, Talsma H, Crom-melin DJA, Hennink WE. 1997. Freeze-drying ofpoly((2-dimethylamino) ethyl methacrylate)-basedgene delivery systems. Pharm Res 14:1838–1841.

20. Talsma H, Cherng JY, Lehrmann H, Kursa M,Ogris M, Hennink WE, Cotton M, Wagner E. 1997.Stabilization of gene delivery systems by freeze-drying. Int J Pharm 157:233–238.

21. Gao X, Huang L. 1996. Potentiation of cationic li-posome-mediated gene delivery by polycations.Biochemistry 35:1027–1036.

22. Li S, Huang L. 1997. In vivo gene transfer via in-travenous administration of cationic lipid-protamine-DNA (LPD) complexes. Gene Ther 4:891–900.

23. Li S, Rizzo MA, Bhattacharya S, Huang L. 1998.Characterization of cationic lipid-protamine-DNA(LPD) complexes for intravenous gene delivery.Gene Ther 5:930–937.

24. Prensky W. 1976. The radioiodination of RNA andDNA to high specific activities. Cell Biol 13:121–152.

25. Li S, Huang L. 1999. Novel lipidic vectors for genetransfer. In: Huang L, Hung M-C, Wagner E, edi-tors. Nonviral vectors for gene therapy. San Diego:Academic Press. p 290–309.

26. Crowe JH, Crowe LM. 1992. Preservation of lipo-somes by freeze-drying. In: Gregoriadis G, editor.Liposome technology, Vol I, Liposome preparationand related techniques. 2nd edition. Boca Raton:CRC Press. p 229–252.

27. Madden TD, Boman N. 1999. Lyophilization of li-posomes. In: Janoff AS, editor. Liposomes: Ratio-nal design. New York: Marcel Dekker, Inc. pp261–282.

28. Anchordoguy TJ, Rudolph AS, Carpenter JF,Crowe JH. 1987. Modes of interaction of cryopro-tectants with membrane phospholipids duringfreezing. Cryobiology 24:324–331.

29. Sternberg B. 1998. Ultrastructural morphology ofcationic liposome–DNA complexes for genetherapy. In: Lasic DD, Papahadjopoulos D, editors.Medical applications of liposomes. New York:Elsevier. pp 395–427.

30. Ross PC, Su H. 1999. Lipoplex size is a major de-terminant of in vitro lipofection efficiency. GeneTher 6:643–650.

31. Liu Y, Mounkes LC, Liggitt HD, Brown CS, Solu-din I, Heath TD, Debs RJ. 1997. Factors influenc-ing the efficiency of cationic liposome-mediated in-travenous gene delivery. Nat Biotechnol 15:167–173.

32. Harrigan PR, Madden TD, Cullis PR. 1990. Protec-tion of liposomes during dehydration or freezing.Chem Phys Lipids 52:139–149.

33. Anchordoquy TJ, Girouard LG, Carpenter JF,Kroll DJ. 1998. Stability of lipid/DNA complexesduring agitation and freeze-thawing. J Pharm Sci87:1046–1051.

34. Liu F, Yang JP, Huang L, Liu D. 1996. New cat-ionic lipid formulations for gene transfer. PharmRes 13:1856–1860.

364 LI ET AL.


Documents

Lyophilization of cationic lipid–protamine–DNA (LPD) complexes