doi: 10.1101/pdb.prot068122Cold Spring Harb Protoc;  Carsten Rudolph and Joseph Rosenecker Transfection of Mammalian Cells In Vitro

Gene Vector Complexes for−Formation of Solid Lipid Nanoparticle (SLN)

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Protocol

Formation of Solid Lipid Nanoparticle (SLN)–Gene VectorComplexes for Transfection of Mammalian Cells In Vitro

Carsten Rudolph and Joseph Rosenecker

Solid lipid nanoparticles (SLNs) offer several technological advantages over standard DNA carrierssuch as cationic lipids or cationic polymers. However, in the absence of endosomolytic agents suchas chloroquine, gene-transfer efficiency mediated by SLN-derived gene vectors consisting of optimizedlipid composition remains lower compared to those achieved with standard transfection agents. Thisprotocol describes the incorporation of a dimeric human immunodeficiency virus type-1 (HIV-1) TATpeptide into SLN gene vectors to increase gene-transfer efficiency. This results in higher transfectionrates than for standard transfection agents in vitro; the ternary SLN–gene vector complexes usuallyresult in transfection levels equal to or higher than those observed with gene vector complexes formu-lated with branched polyethylenimine (PEI) 25 kDa. One significant advantage of using this method isthe low cytotoxicity of the SLN gene vectors. The application of the gene-transfer technique is limitedto relatively low plasmid DNA (pDNA) concentrations of the resulting complexes (10 µg/mL). Athigher concentrations, the particles tend to aggregate and precipitate. Therefore, their use for invivo application, which generally requires high pDNA concentrations, is limited.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s EnvironmentalHealth and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Reagents

Cell line to be transfected

Cell growth medium (serum-free), as appropriate for cell line of interest

Cetylpalmitate (Henkel, Düsseldorf, Germany) (to be used as the matrix lipid)

DOTAP (1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane; Sigma-Aldrich) (to be used as thecationic lipid)

Fetal calf serum (FCS)

Gentamycin (Invitrogen)

HEPES-buffered saline (HBS; 150 mM NaCl, 10 mM HEPES [pH 7.4])

Penicillin/streptomycin (Gibco/Invitrogen 15140–122)

Plasmid of interest (pDNA)

Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (ed. Friedmann and Rossi). CSHL Press, Cold Spring Harbor,NY, USA, 2007.

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Reagents for synthesizing the TAT2 peptide, C(YGRKKRRQRRRG)2 (containing the arginine-richmotif of the HIV-1 TAT protein)

Span 85 (ICI Surfactants)

Tween 80 (ICI Surfactants)

Equipment

Automatic synthesizer (Applied Biosystems 431A)

Cell culture plates (24 well)

High-pressure homogenizer (EmulsiFlex-B3; Avestin Inc.)

High-speed stirrer (Ultra Turrax T25; Janke & Kunkel)

Incubator (37˚C, 5% CO2)

Mass spectrometer

Reversed-phase HPLC

METHOD

Preparation of SLNs and the TAT2 Peptide

1. Prepare SLNs by hot high-pressure homogenization as described previously (Müller et al.2000a).

i. Heat the solid lipids to �10˚C above their melting points.

ii. Mix the surfactants Tween 80 and Span 85 in a 7:3 ratio.

iii. Combine the surfactant mix (2% w/w) with 1% (w/w) cationic DOTAP in a hot aqueoussolution.

iv. Combine moltenmatrix lipid (cetylpalmitate, 4%w/w) and the hot aqueous solution of sur-factants and DOTAP into a preemulsion by stirring with a high-speed stirrer for 1 min.

v. Homogenize batches of SLNs containing the DOTAP at 85˚C at a pressure of 480 bar andfour homogenization cycles using a high-pressure homogenizer.

2. Prepare the TAT2 peptide.

i. Synthesize the peptide on an automatic synthesizer according to a standard Fmoc protocol.

ii. Purify the peptide by reversed-phase HPLC.

iii. Analyze by mass spectroscopy.The free sulfhydryl groups are modified by the dithiodipyridin reaction (Plank et al. 1999). This modi-fication is important to avoid disulfide formation of the TAT2 peptide in aqueous solution.

Preparation of SLN–Gene Vector Complexes

A schematic drawing of the ternary gene vector formulation is shown in Figure 1.

3. For each well of a 24-well plate, prepare the following solutions.

i. Dilute 1 µg of pDNA in HBS to a final volume of 50 µL.

ii. Dilute 0.65 µg of TAT2 peptide (prepared in Step 2) in HBS to a final volume of 50 µL.

iii. Dilute 2.5 µg of SLNs (prepared in Step 1) in HBS to a final volume of 50 µL.

4. To prepare the core complex, add the pDNA solution to the TAT2 solution. Mix vigorously bypipetting up and down 10 times. Incubate for 10 min at room temperature.

5. To prepare the ternary complex, add the SLN solution to the core complex. Mix by pipetting upand down 10 times. Incubate for 10 min at room temperature.

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In Vitro Transfection

6. One day before transfection, seed the cells to be transfected in a 24-well plate in cell growthmediumsupplementedwith FCS. (Cells should be 60–70%confluent at the timeof transfection.)

7. Aspirate the complete cell growth medium from each well. Replace with 850 µL of serum-freemedium before transfection.

8. Add 150 µL of SLN–gene vector ternary complex solution (from Step 5) to the cells. Incubatefor 4 h at 37˚C in a 5% CO2 atmosphere.

9. Aspirate the transfection medium. Replace with cell growth medium supplemented with 10%FCS and 0.1% (v/v) penicillin/streptomycin and 0.5% (v/v) gentamycin.

10. Measure the gene-transfer efficiency at the desired timepoint after transfection.

DISCUSSION

SLNs were invented at the beginning of the 1990s and were produced either by high-pressure hom-ogenization or by microemulsion techniques. From the point of view of production and regulatoryaspects, high-pressure homogenization is the method of choice. SLNs consist of a solid matrix.They are comparable with parenteral emulsions, except that in SLN formulations, the liquid lipid(oil) is replaced by solid lipid. Cationic SLNs condense DNA into nanometric colloidal particlescapable of transfecting mammalian cells in vitro (Olbrich et al. 2001; Tabatt et al. 2004). In thesestudies, cationic SLNs were produced by hot homogenization using either Compritol ATO 888 (amixture of mono-, di-, and triglycerides of behenic acid) or paraffin as matrix lipid, a mixture ofTween 80 and Span 85 as tenside and either EQ1 (N,N-di-(β-steaorylethyl)-N,N-dimethylammoniumchloride) or cetylpyridinium chloride as the charge carrier. More detailed analyses of cationic lipid andmatrix lipid composition of SLNs revealed that plasmid DNA binding, cytotoxicity, and transfectionefficiency were dependent on the structure of the cationic lipid and the matrix lipid. Whereas SLNsmade from two-tailed cationic lipids were well tolerated in cell culture, SLNs made from one-tailedcationic detergents were highly toxic. These studies revealed that optimal SLN formulations forgene transfer were made from DOTAP as the cationic lipid and cetylpalmitate as the matrix lipid.

Compared with standard DNA carriers such as cationic lipids or cationic polymers, SLNs offerseveral technological advantages. They are relatively easy to produce without the use of organic sol-vents (Mehnert and Mader 2001), and large-scale production is possible with qualified productionlines (Müller et al. 2000a; Dingler and Gohla 2002). They show good stability during the long-termstorage (Freitas and Müller 1999) and are amenable to both steam sterilization (Schwarz et al.1994) and lyophilization (Schwarz and Mehnert 1997). Perhaps most importantly, as substancesthat are generally recognized as safe (Müller et al. 2000b), they are less toxic (Olbrich et al. 2001)

FIGURE 1. Formulation of ternary SLN–gene vectors. The desired plasmidDNA is added to the TAT2 peptide solution, mixed, and incubated for 10min at room temperature before the solution containing the SLNs is added.

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Forming SLN-Gene Vector Complexes for Transfection

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than cationic polymers such as PEI (Bragonzi et al. 2000; Gebhart and Kabanov 2001). However, inthe absence of endosomolytic agents such as chloroquine, gene-transfer efficiency mediated by SLN-derived gene vectors (even when the lipid composition is optimized) remains lower than thoseobserved with standard transfection agents such as PEI 25 kDa (Olbrich et al. 2001; Tabatt et al. 2004).

Subsequent studies showed that precompaction of DNAwith oligomers of the HIV-1 TAT peptidefor the formulation of gene vector complexes led to an increase of up to two orders of magnitude ingene-transfer efficiency (Rudolph et al. 2003). The dimeric TAT peptide was found to be most effi-cient. This effect was related to the unique features of the HIV-1 TAT peptide which represents aprotein transduction domain (Frankel and Pabo 1988; Fawell et al. 1994) and a nuclear localizationsequence (Truant and Cullen 1999). This protocol describes the formulation of ternary gene vectorcomplexes consisting of DNA precompacted with a dimeric TAT peptide (TAT2), which is then com-pleted by the addition of a cationic SLN gene carrier. When the plasmid DNA is first complexed withthe TAT2 peptide under the given conditions (corresponding to a charge ratio of ±1) the resultingintermediate complexes have a zeta potential of approximately –20 mV. The zeta potentials aremeasured electrophoretically (ZetaPALS/Zeta Potential Analyzer; Brookhaven Instruments Corpor-ation, Austria). The following settings were used: 10 subrun measurements/sample; viscosity forH2O 0.89 cP; beam mode F(Ka) = 1.50 (Smoluchowsky); temperature 25˚C. These preformed nega-tively charged TAT2–plasmid DNA (pDNA) gene vector complexes allow the binding of positivelycharged SLNs to their surface through electrostatic interaction. The formulation method apparentlyresults in a shell-like ternary complex with the TAT2 peptide bound to the plasmid DNA in the core ofthe complex and a layer of SLN at the periphery of the complex. Therefore, our experience is that thepH of the solutions is important to result in negatively charged TAT2–pDNA complexes. Such genevectors increase gene-transfer efficiency, resulting in higher transfection rates than for standard trans-fection agents in vitro, but with lower toxicity.

ACKNOWLEDGMENTS

The authors would like to acknowledge Dr. Kerstin Tabatt and Prof. Dr. R.H. Müller for providing theSLN formulations and the Deutsche Forschungsgemeinschaft Ro994/2–1 for funding of the work.

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