[Methods in Enzymology] Nanomedicine - Infectious Diseases, Immunotherapy, Diagnostics,...

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Lipopolyplexes as Nanomedicines for

Therapeutic Gene Delivery

Leire Garcıa,* Koldo Urbiola,* Nejat Duzgunes,† and

Conchita Tros de Ilarduya*

Contents

1. In

in

076

rtmlonrtmran

troduction

Enzymology, Volume 509 # 2012

-6879, DOI: 10.1016/B978-0-12-391858-1.00016-2 All rig

ent of Pharmacy and Pharmaceutical Technology, School of Pharmacy, Universitya, Spainent of Biomedical Sciences, University of the Pacific Arthur A. Dugoni School ocisco, California, USA

Else

hts

of

f D

328

2. P
rinciple of the Method 329
3. E
xperimental Procedures 329
3
.1. M aterials 329
3
.2. C ell culture 330
3
.3. P reparation of lipopolyplexes 330
3
.4. D NA/PEI condensation assay 331
3
.5. P article size and zeta potential measurements 331
3
.6. In vitro transfection activity 331
4. A
pplication of Lipopolyplexes 332
4
.1. C haracterization of complexes 332
4
.2. In vitro transfection activity measurements 334
5. C
oncluding Remarks 336
Ackn
owledgments 336
Refe
rences 337
Abstract

We describe an efficient, nonviral gene transfer system that employs polyethy-

lenimine (PEI 800, 25, 22 kDa), and 1,2-dioleoyl-3-(trimethylammonium) pro-

pane (DOTAP) and cholesterol (Chol) as lipids (lipopolyplex), at three different

lipid/DNA molar ratios (2/1, 5/1, and 17/1), employing five different formula-

tion strategies. PEIs of 800, 25, and 22 kDa are highly effective in condensing

plasmid DNA, leading to a complete condensation at N/P (+/�) ratios above 4.

Increasing the molar ratio lipid/DNA in the complex results in higher positive

values of the zeta potential, while the particle size increases in some protocols,

but not in others. PEI of molecular weight 800 kDa used in the formulation of

vier Inc.

reserved.

Navarra,

entistry,

327

328 Leire Garcıa et al.

lipopolyplexes results in bigger particles compared to that obtained with the

smaller PEI species. Transfection activity is measured using pCMVLuc expressing

luciferase is maximal by using strategies 3 and 4 and an N/P molar ratio of 17/1.

These complexes have a high efficiency of gene delivery to liver cancer cells,

even in the presence of a high serum concentration. Complexes formed with

linear PEI are more effective than lipopolyplexes containing branched PEI. The

ternary complexes are much more efficient than conventional lipoplexes (cat-

ionic lipid and DNA) and polyplexes (cationic polymer and DNA). The same

behavior is observed for complexes prepared with the therapeutic gene

pCMVIL-12 expressing interleukin-12.

1. Introduction

Gene therapy focuses on the therapeutic use of genes delivered tocells, and may lead to advances in the treatment of numerous diseasesincluding cancer and genetic diseases (Drew and Martin, 1999). Althoughprogress has been made in identifying target structures for cancer genetherapy, actual therapy is limited by the lack of a safe and efficient genedelivery system. Currently, one of the primary objectives of gene therapy isthe development of efficient, nontoxic gene carriers that can effectivelydeliver therapeutic genetic material into specific cell types, including cancercells.

The two main gene carrier systems that have been utilized in genetherapy are viral vectors and nonviral delivery systems (Pedroso de Limaet al., 2001; Tros de Ilarduya et al., 2010). Viral vectors, including retro-viruses, adenoviruses, and adeno-associated viruses, have a high efficiency ofgene delivery. Because of serious safety risks with viral vectors that havebecome apparent in the past few years, however, their utility is beingreappraised. Furthermore, the addition of targeting ligands on the surfaceof viral vectors to transfect specific cell types is problematic. Because of theseconcerns, nonviral vectors are emerging as a viable alternative. Nonviralsystems present significantly lower safety risks, and can be produced easilyand inexpensively in large quantities. A major disadvantage of nonviral genecarriers is their low transfection efficiency, especially in the presence ofserum.

Strategies such as particle bombardment (Mahato et al., 1999), ultra-sound transfection (Newman et al., 2001), or the application of nakedDNA (Shi et al., 2002) have been used to deliver genes. Cationic poly-mers and lipids are the most widely used vectors in nonviral gene andoligonucleotide delivery. Some liposomal formulations have serious lim-itations because of their low transfection efficiency and cytotoxicity (Gaoand Huang, 1995; Meyer et al., 1998). Polycationic polymers can

Lipopolymeric Nanocarriers for Gene Delivery 329

condense DNA, which is an advantage in gene transfer (Goldman et al.,1997; Sorgi et al., 1997). Polyethylenimine (PEI) has been used success-fully for in vitro and in vivo gene delivery (Boussif et al., 1995; Lungwitzet al., 2005). It can condense and protect DNA, facilitate binding to thecell surface, trigger endocytosis, and mediate the release DNA/lipid com-plexes from endosomes into the cytoplasm as a result of the “proton-sponge” effect (Behr, 1994). It can enter the nucleus (Godbey et al., 1999)and thus accelerate DNA entry into the nucleus (Pollard et al., 1998).Although cationic liposomes can deliver DNA into the cytosol followingendocytosis, the entry of the DNA into the nucleus is inefficient (Fisheret al., 2000; Harashima et al., 2001).

2. Principle of the Method

Lipopolyplexes (i.e., a ternary complexes of cationic liposomes, cat-ionic polymer, and DNA) constitute a second generation of nonviral genecarriers that can improve gene transfer compared to the first generationcationic liposome-DNA complexes (lipoplexes) (Gao and Huang, 1996;Lampleta et al., 2003; Matsura et al., 2003; Pelisek et al., 2006; Sorgi et al.,1997; Whitmore et al., 1999; Yamazaki et al., 2000).

Because of our interest in developing new methods to prepare lipo-polyplexes as nanomedicines for therapeutic gene delivery, we have eval-uated different parameters to optimize the formulation to achieve hightransfection activity, including the protocol of preparation, the lipid/DNA molar ratio, and the molecular weight and type of PEI. We hypothe-sized that the association of PEI with cationic liposomes (lipopolyplexes)would increase luciferase expression compared to lipoplexes (cationic lipidand DNA) and polyplexes (cationic polymer and DNA) alone (Garcıaet al., 2007).

3. Experimental Procedures

3.1. Materials

The cationic lipid, 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP),and cholesterol (Chol) are obtained from Avanti Polar Lipids (Alabaster,AL, USA). PEI 800 (MW 800 kDa, branched) is available from Fluka(Steinheim, Germany), PEI 25 (MW 25 kDa, branched) from Aldrich(Madrid, Spain), and linear PEI (MW 22 kDa, ExGenÒ 500) from Quimi-granel (Madrid, Spain). The plasmids, pCMVLuc (VR-1216) (6934 bps)


(Clontech, Palo Alto, CA, USA) and pCMV100-IL-12 (5500 bps) (kindlyprovided byDr. ChenQian, University of Navarra), encoding luciferase andinterleukin-12 (IL-12), respectively, are used for these studies.

3.2. Cell culture

HepG2 human hepatoblastoma cells (American Type Culture Collection,Rockville, MD, USA) are maintained at 37 �C under a 5% CO2 atmo-sphere in Dulbecco’s modified Eagle’s medium high glucose, supplementedwith 10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin(100 units/ml), streptomycin (100 mg/ml), and L-glutamine (4 mM; GibcoBRL Life Technologies). Cells are passaged 1:10 following trypsinizationonce a week.

3.3. Preparation of lipopolyplexes

Lipopolyplexes are prepared with plasmid DNA and B-PEI (branched, 800or 25 kDa) or L-PEI (linear, 22 kDa) at an N/P ratio of 4. Differentamounts of lipids are added to prepare complexes at molar ratios of totallipid/DNA of 2/1, 5/1, and 17/1. The final DNA concentration in thelipopolyplex suspension is 25 mg/ml in a total volume of 3 ml.

Lipopolyplexes are formulated using the following strategies:

Strategy 1 involves drying a chloroform solution of the lipids, DOTAP/Chol (1:0.9 molar ratio), by rotary evaporation, and then hydrating thefilm with the polyplexes (PEI/DNA) (N/P ratio of 4).

Strategies 2 and 3 are carried out by drying a chloroform solution of thelipids (DOTAP/Chol) and PEI by rotary evaporation. In Strategy 2,the film is hydrated with water, and then the plasmid is added. InStrategy 3, the mixture of lipids and PEI are hydrated with the plasmidsolution.

In strategies 4 and 5, the polyplexes, PEI/DNA, are generated at an N/Pratio of 4 and, after a 15 min incubation, different amounts of pre-formed cationic liposomes are added, and to obtain complexes atvarious lipid/DNA N/P ratios (2/1, 5/1, and 17/1). These strategiesdiffer from each other only in the order of addition of plasmid to obtainthe polyplexes: PEI added to DNA (Strategy 4) or DNA added to PEI(Strategy 5).

The prepared complexes are extruded through polycarbonate mem-branes (100 nm pore diameter), using a Liposofast device (Avestin, Tor-onto, ON, Canada) to obtain particles with a uniform size distribution.


3.4. DNA/PEI condensation assay

The binding of PEI to DNA is investigated using ethidium bromide, whosefluorescence is enhanced greatly upon binding to DNA, and quenchedwhen it is displaced by the condensation of the DNA structure (Tros deIlarduya et al., 2002). The assays are carried out in 96-well plates in 10% (w/v) glucose, 10 mM HEPES buffer (pH 7.4). DNA (0.6 mg) is mixed with1.2 mg of ethidium bromide, then increasing amounts of PEI are added tothe wells and incubated for 10 min in the dark. Fluorescence is read in an LS50 spectrofluorimeter (Perkin-Elmer, Mountain View, CA, USA), at exci-tation and emissionwavelengths of 520 and 600 nm, respectively. The relativefluorescence values are evaluated as follows: Fr¼ (Fobs–Fe)�100/(F0–Fe),where Fr is the relative fluorescence, Fobs is the measured fluorescence, Fe isthe fluorescence of ethidium bromide in the absence of DNA, and F0 is theinitial fluorescence in the absence of the polycation.

3.5. Particle size and zeta potential measurements

The particle size of the complexes is measured by dynamic light scattering,and the overall charge of the particles by zeta potential measurements, usinga Zeta Nano Series particle analyzer (Malvern Instruments, Spain). Aliquotsof the complexes are diluted in distilled water, and are measured at leastthree times immediately after the preparation of the complexes.

3.6. In vitro transfection activity

The cells are seeded in medium in 48-well culture plates (Iwaki Microplate,Japan) and incubated for 24 h at 37 �C in 5% CO2. The medium is removedand 0.2 ml of the complexes (containing 1 mg of plasmid) and 0.3 ml of FBSare added to each well. The complexes are thus incubated with the cellsfor 4 h in the presence of 60% FBS. The complexes are then removed, andmedium containing 10% FBS is added. The cells are further incubatedfor 48 h; then they are washed with phosphate-buffered saline and lysedwith 100 ml of Reporter Lysis Buffer (Promega, Madison, WI, USA). Themixture is incubated at room temperature for 10 min, and then frozen andthawed twice. The cell lysate is centrifuged for 2 min at 12000� g to pelletcellular debris. Twenty microliters of the supernatant are assayed for totalluciferase activity, using the Luciferase Assay Reagent (Promega), accordingto the manufacturer’s instructions. A luminometer (Sirius-2, BertholdDetection Systems, Innogenetics, Diagnostica y Terapeutica, Barcelona,Spain) is used to measure luciferase activity. The protein content of thelysates is measured by the DC protein Assay Reagent (Bio-Rad, Hercules,CA, USA), with bovine serum albumin as the standard. The data are


expressed as nanograms of luciferase (based on a standard curve for luciferaseactivity) per milligram of protein. IL-12 levels are determined by an ELISAkit for murine IL-12p70 (BD OptEIA ELISA sets, Pharmingen, San Diego,CA, USA), according to the manufacturer’s instructions.

4. Application of Lipopolyplexes

4.1. Characterization of complexes

We prepared lipopolyplexes according to each of the five strategies by usingPEI 800, 25, and 22 kDa, and DOTAP and cholesterol at lipid/DNA N/Pratios of 2/1, 5/1, and 17/1, as described above.

To prepare complexes in which DNA was condensed completely byPEI, the corresponding condensation studies were performed, by measuringthe decrease in the ethidium bromide fluorescence. As shown in Fig. 16.1,the fluorescence decreased with increasing N/P ratio (Garcıa et al., 2007).The fluorescence reached a plateau at N/P ratios of 4–10, with the DNAbeing condensed to less than 10% of the uncomplexed DNA, suggestingthat DNA is condensed completely at these ratios. The same behavior wasobserved with all three polymers of PEI. Increasing amounts of lipid addedto polyplexes at an N/P ratio of 4 did not show any additional effect on thecondensation of the plasmid.

Complexes were characterized in terms of particle size and zeta poten-tial. At a lipid/DNA ratio of 2/1, the largest lipopolyplexes were thoseprepared using Strategy 2 (281 nm), whereas at the higher ratios, Strategy 3

0

10

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40

50

60

70

80

90

100

0 2 4 6 8 10N/P ratio

% F

luor

esce

nce

PEI 800 kDa

PEI 25 kDa

PEI 22 kDa

Figure 16.1 PEI/DNA condensation assay. DNA condensation measured as adecrease in the fluorescence of EtBr added to polyplexes (From Garcıa et al., 2007).

Table 16.1 Particle size of lipopolyplexes, prepared with PEI 800 kDa, as a functionof the protocol of formulation at the indicated lipid/DNA molar ratios (From Garcıaet al., 2007)

Particle size (nm)

Molar ratio

(lipid/DNA) Strategy 1 Strategy 2 Strategy 3 Strategy 4 Strategy 5

2/1 188�8 281�10 229�14 157�3 124�2

5/1 187�6 232�8 356�11 181�4 183�1

17/1 205�4 211�20 295�2 214�8 201�8

Table 16.2 Zeta potential of lipopolyplexes, prepared with PEI 800 kDa, as a functionof the protocol of formulation at the indicated lipid/DNA molar ratios (From Garcıaet al., 2007)

Zeta potential (mV)

Molar ratio

(lipid/DNA) Strategy 1 Strategy 2 Strategy 3 Strategy 4 Strategy 5

2/1 55�3 34�4 35�1 57�2 68�1

5/1 57�1 48�1 54�3 68�1 71�3

17/1 69�2 49�2 59�2 69�1 72�1


produced the largest lipopolyplexes (356 and 295 nm for 5/1 and 17/1ratios, respectively; Table 16.1) (Garcıa et al., 2007). At the 2/1 lipid/DNAratio, the smallest particle size (124 nm) was obtained using Strategy 5. Mostof the other complexes had average particle sizes of about 200 nm. Regard-ing the zeta potential measurements, at the 2/1 lipid/DNA ratio, thesmallest lipopolyplexes prepared by Strategy 5 also had the highest netpositive charge (zeta potential¼68 mV), whereas the largest ones (Strategy2) had the lowest zeta potential (34 mV; Table 16.2). All the preparationstrategies resulted in net positively charged complexes. Increasing thelipid/DNA ratio appeared to increase the zeta potential for lipopolyplexesprepared by each strategy (Table 16.2).

Complexes were prepared by strategy 4 at an N/P ratio of 17/1 to studythe influence of the molecular weight and type of PEI on the particle sizeand zeta potential of lipopolyplexes. Table 16.3 shows that complexesprepared with PEI 800 of high molecular weight were bigger in particlesize compared to lipopolyplexes containing PEI 25 or PEI 22. No differ-ences in the particle size were observed between lipopolyplexes preparedwith branched (25 kDa) or linear (22 kDa) PEI of similar molecular weight(Garcıa et al., 2007). Complexes prepared with the branched PEIs had aslightly higher zeta potential compared to the linear PEI.

Table 16.3 Influence of molecular weight and type of PEI on the particle size andzeta potential of lipopolyplexes (From Garcıa et al., 2007)

Particle size (nm) Zeta potential (mV)

PEI 800 branched 214�8 69�1

PEI 25 branched 134�4 60�4

PEI 22 linear 147�2 51�2

Complexes were prepared by protocol 4 at a lipid/DNAmolar ratio of 17/1. Results are expressed as themean�SD of three independent experiments.


4.2. In vitro transfection activity measurements

We examined gene expression mediated by lipopolyplexes as a function ofthe strategy of formulation and the lipid/DNA molar ratio of the com-plexes, in the presence of 60% FBS. As shown in Fig. 16.2, lipopolyplexesformulated with strategies 3 and 4 were more effective in transfectingHepG2 cells than complexes generated with the other strategies, 1, 2, and5, independently of the lipid/DNA molar ratio used to prepare them(Garcıa et al., 2007). The highest expression was achieved with complexesprepared by Strategy 4 at a molar ratio lipid/DNA of 17/1, which showed a16-, 28-, 1.3-, and 25-fold increased transfection compared to lipopoly-plexes formulated by the strategies 1, 2, 3, and 5, respectively, preparedat the same molar ratio. Transfection activity increased by increasing the

NakedDNA

ng luc

ifer

ase/

mg

prot

ein

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5

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25

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35

40

Polyplexes LPP(Strategy 1)

LPP(Strategy 2)

LPP(Strategy 3)

LPP(Strategy 4)

LPP(Strategy 5)

Lipoplexes

Molar ratio 2/1 Molar ratio 5/1 Molar ratio 17/1

Figure 16.2 Transfection activity in HepG2 cells by polyplexes, lipoplexes, and lipo-polyplexes (LPP) in the presence of 60% FBS. Complexes were formulatedwith branchedPEI of 800 kDa with 1 mg of pCMVLuc. The data represent the mean�SD of three wellsand are representative of three independent experiments (From Garcıa et al., 2007).


lipid/DNAmolar ratio in the lipopolyplexes, independently of the protocolused to prepare the complexes. The transfection activity increased by 1.7-and 33-fold with complexes prepared at the 17/1 molar ratio compared tolipopolyplexes at the 5/1 and 2/1 molar ratios, respectively, by usingstrategy 4. Lipopolyplexes prepared with strategies 3 and 4 showed muchhigher transfection activity than conventional polyplexes or lipoplexes,particularly at lipid/DNA ratios of 5/1 and 17/1. Naked DNA did notinduce any measurable luciferase expression.

We investigated the effect of the molecular weight and the type ofpolymer on polyplex- and lipopolyplex-mediated transfection of HepG2cells. For these experiments, lipopolyplexes were formulated using Strategy 4at a lipid/DNA molar ratio of 17/1. No significant differences were found inthe levels of gene expression by using PEI 800 or 25 kDa (Fig. 16.3). Com-plexes formed with linear PEI (22 kDa) were slightly more effective thanlipopolyplexes containing branched PEI (800 or 25 kDa). Lipopolyplexesmediated much higher transfection activity than conventional polyplexes,with lipopolyplexes prepared with PEI 22 kDa resulting in 26 times higherluciferase expression than the polyplexes prepared with the same polymer.

We also prepared lipopolyplexes were prepared in the presence ofpCMVIL-12 to examine the ability of liver cancer cells to express a poten-tially therapeutic gene. Lipopolyplexes prepared with linear PEI 22 kDa

0Polyplexes LPP (Strategy 4)

5

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20

25

30

35

ng luc

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ase/

mg

prot

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40

45

50

55

60

65PEI 800 kDa PEI 25 kDa PEI 22 kDa

Figure 16.3 Transfection activity by polyplexes and lipopolyplexes (LPP) in HepG2cells in the presence of 60% FBS. Complexes were prepared with different types of PEI,at a lipid/DNA molar ratio of 17/1, and contained 1 mg of pCMVLuc. The datarepresent the mean�SD of three wells and are representative of three independentexperiments (From Garcıa et al., 2007).

Lipopolyplexes0

10

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L-1

2/m

l 40

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60 800 kDa 25 kDa 22 kDa

Figure 16.4 IL-12 gene expression following transfection of HepG2 cells by lipopoly-plexes (LPP), in the presence of 60% FBS. Complexes were formulated by protocol 4 ata lipid/DNAmolar ratio of 17/1 and contained 1 mg of pCMVIL-12. The data representthe mean�SD of three wells and are representative of three independent experiments(From Garcıa et al., 2007).


mediated higher IL-12 expression than lipopolyplexes containing PEI 800 or25 kDa (Fig. 16.4), in general agreement with the results obtained with theplasmid encoding luciferase (Garcıa et al., 2007). Transfection activity oflipoplexes and polyplexes mediated lower levels of transgene expressioncompared to that observed with lipopolyplexes.

5. Concluding Remarks

We have developed an efficient gene delivery vehicle by combiningPEI and DOTAP/Chol liposomes (Garcıa et al., 2007). The advantages ofthese complexes include (i) small particle size to potentially improve trans-fection efficiency in vivo, (ii) reproducibility of transfection efficacy, (iii)efficient transfection of liver cancer cells in the presence of a high concen-tration of serum, and (iv) stability of the complexes. Since both PEI andliposomes can be modified easily with various ligands, these lipopolyplexesmight be useful in the design of targeted carriers specific for certain cell typesand the delivery of therapeutic genes. Future studies will investigate thesuitability of this nonviral vector for gene transfer in vivo.

ACKNOWLEDGMENTS

This work was financially supported by the University of Navarra Foundation (FUN) andthe Caja de Ahorros de Navarra (CAN).


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