a dimethylmaleic acid–melittin-polylysine conjugate with reduced toxicity, ph-triggered...

THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2007; 9: 797–805.Published online 13 July 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1075

A dimethylmaleic acid–melittin-polylysineconjugate with reduced toxicity, pH-triggeredendosomolytic activity and enhanced gene transferpotential

Martin MeyerArkadi ZintchenkoManfred OgrisErnst Wagner*

Pharmaceutical Biotechnology,Department of Pharmacy,Ludwig-Maximilians-Universitat,Butenandtstr. 5-13, D-81377Munich, Germany

*Correspondence to: Ernst Wagner,PharmaceuticalBiology-Biotechnology, Departmentof Pharmacy,Ludwig-Maximilians-Universitat,Butenandtstr. 5-13, D-81377Munich, Germany. E-mail:[email protected]

Received: 22 February 2007Revised: 22 May 2007Accepted: 23 May 2007

Abstract

Background Poor endosomal release is one major barrier of gene delivery.Endosomolytic polyethylenimine-melittin conjugates have shown to enhancegene transfer efficiency; however, cytotoxicity due to their general membrane-destabilizing properties limits their application. To overcome this drawbackwe grafted a polycation with a masked pH-responsive melittin derivate andinvestigated lytic activity, gene transfer efficiency and cytotoxicity of theresulting conjugate.

Methods Melittin (Mel) was modified with dimethylmaleic anhydride(DMMAn) and covalently coupled to poly-L-lysine (PLL). The membranelytic activity was analyzed after incubation at neutral or endosomal pH.PLL-DMMAn-Mel polyplexes were generated in HEPES-buffered glucoseand tested in transfection experiments using luciferase as reporter gene.Cellular cytotoxicity was analyzed by measurement of membrane integrityand metabolic activity.

Results Covalent attachment of DMMAn-modified melittin to PLL resultedin a pH-responsive conjugate. No lytic activity was observed at neutral pH;after acidic cleavage of the protecting groups at pH 5 lytic activity wasregained. Acute toxicity was greatly reduced (as compared to PLL-Mel oreven unmodified PLL) and high gene expression levels (up to 1800-foldhigher than unmodified PLL) were obtained.

Conclusions Modification of the polycationic carrier PLL with DMMAn-masked melittin not only enhances gene transfer efficiency, but also stronglyreduces the acute toxicity of melittin and PLL. Hence this modification mightbe useful for optimizing polycationic gene carriers. Copyright 2007 JohnWiley & Sons, Ltd.

Keywords melittin; nonviral gene transfer; pH-responsive; synthetic virus

Introduction

Relatively low transfection efficiency of available nonviral gene carri-ers limits their in vivo use. A crucial bottleneck in gene delivery ispoor endosomal release after cellular internalization of the gene car-rier. It is essential to overcome this barrier to avoid degradation

Copyright 2007 John Wiley & Sons, Ltd.

798 M. Meyer et al.

in the endolysosomal compartment. Transfection effi-ciency can be improved by incorporation of endosomolyticdomains into the vector. Several endosomolytic peptides,which were derived from viruses [1,2] toxins [3,4], orsynthetically designed [5], have been applied for thesepurposes [6].

Melittin, a 26 amino acid peptide, displays a particularlystrong lytic activity and enhances the transfectionefficiency of polycations [7–10] and lipids [11]. Onemajor disadvantage of melittin is a high lytic activityat neutral pH, which might lead to toxic side effectsbefore internalization into cells. After internalization ofthe gene carrier the endosome is acidified [12]. Viruseslike adenovirus use this acidification process as a triggerto expose endosomolytic domains, which facilitates therelease of the virus into the cytosol [13]. Hence, apronounced lytic activity under acidic conditions andreduced activity at neutral pH would be favorable alsoin case of melittin. Murata et al. could show that lyticactivity is minimized when the lysines in melittin andits terminal amino function were irreversibly acylatedwith succinic anhydride [14]. The concept of a reversibleacylation of melittin through a dimethylmaleic anhydridederivative was first presented by Rozema et al. [15] andgenerated a lytic activity triggered only upon acidification.Simultaneous co-delivery of the modified peptide and theoligonucleotides enhanced cytosolic drug delivery in vitro[15].

Since melittin does not stably associate with DNA orDNA/polycation complexes [1,16], the peptide has tobe coupled covalently to the gene delivery carrier foroptimal co-delivery. Therefore, the aim of this study was tosynthesize a conjugate on the basis of a reversibly acylatedmelittin with the favored bioresponsive lytic activity.We have compared the bioresponsive conjugate with anunmodified melittin conjugate in terms of membrane-destabilizing effects, gene transfer efficiency in differentpolyplex compositions and cytotoxicity.

Materials and methods

Chemicals and reagents

Poly-L-lysine hydrobromide with an average molec-ular weight of 55 kDa (PLL), succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 2,3-dimethyl-maleicanhydride (DMMAn) were purchased from Sigma-Aldrich (Munich, Germany). Cysteine-modified melittin(Mel) was obtained from IRIS Biotech GmbH (Mark-tredwitz, Germany) and had the sequence CIGA VLKVLTTG LPAL ISWI KRKR QQ (all-(D) configuration). All-(D)stereochemistry was used because it is non-immunogenicwhile being as lytic as the natural peptide [7]. PLL-PEGwas synthesized and purified as previously described [17](with a molar ratio of 20 kDa PEG to 55 kDa PLL of1 : 1). PLL-transferrin (PLL-Tf) was synthesized as previ-ously described [18] (with a molar ratio of transferrinto 52 kDa PLL of 0.7 : 1). Plasmid pCMVLuc (Photinus

pyralis luciferase under control of the cytomegalovirus(CMV) enhancer/promoter), described previously [19],was produced with the Qiagen Plasmid Giga kit (Qia-gen, Hilden, Germany) according to the manufacturer’srecommendations.

Conjugate synthesis

Synthesis of 3-(2-pyridyldithio)propionate-modified PLLPLL (1 µmol) in 0.8 ml buffer (0.5 M NaCl, 20 mMHEPES, pH 7.4) was mixed with SPDP (20 µmol) dis-solved in 200 µl dimethyl sulfoxide (DMSO). After 2 hat room temperature (RT) PLL with pyridyldithiopropi-onate linkers (PLL-PDP) was purified by gel filtrationusing an Akta Basic high-performance liquid chromatog-raphy (HPLC) system (Amersham Biosciences, Freiburg,Germany) equipped with a Sephadex G-25 superfine HR10/30 column (Pharmacia Biotech, Uppsala, Sweden)equilibrated in 500 mM NaCl, 20 mM HEPES, pH 7.4;the flow rate was 0.5 ml/min. The void fractions con-taining PLL were pooled, aliquots were snap frozen inliquid nitrogen and stored at −80 ◦C. PLL content of thefractions was measured by trinitrobenzenesulfonic acid(TNBS) assay at 405 nm as described [20]. The degreeof modification with the dithiopyridine linker was deter-mined spectrophotometrically at 343 nm by release ofpyridine-2-thione (molar absorptivity = 8080 M−1 cm−1)after reduction of an aliquot with excess of dithiothre-itol. For the synthesis of PLL-Mel and PLL-DMMAn-Mel,the same PLL-PDP batch was used with a molar ratio ofPLL/PDP of approximately 1 : 13.

Synthesis of PLL-Mel conjugatesMel peptide (1.38 µmol) in all-(D) configuration wasdissolved in 400 µl of 0.5 M NaCl, 20 mM HEPES, pH7.4, and mixed with 1000 µl PLL-PDP (71 nmol PLL,0.93 µmol PDP) diluted in the same buffer under argon.After 3 h at RT released thiopyridone was measured at343 nm to determine the extent of the reaction. Melittin-PLL conjugates were purified on the Akta Basic HPLCsystem equipped with a Superdex 75 HR 10/30 columnequilibrated in 500 mM NaCl, 20 mM HEPES, pH 7.4. Theflow rate was 0.5 ml/min. The void fractions containingPLL-Mel were pooled, aliquots were snap frozen in liquidnitrogen and stored at −80 ◦C. The PLL content of the PLL-Mel conjugate was determined by TNBS assay at 405 nm[20]. The concentration of melittin in the conjugates wasmeasured by absorption at 280 nm (molar absorptivity ofmelittin = 5570 M−1 cm−1 [21]).

Synthesis of the PLL-DMMAn-Mel conjugateMel peptide (1.38 µmol) was dissolved in 400 µl of100 mM HEPES and 125 mM NaOH and mixed with1000 µl ethanol containing 15.8 µmol DMMAn by rapidvortexing under argon for 0.5 h following concentrationand purification via ultrafiltration (Vivascience, Vivaspin2, MWCO 2000 HY). After modification no amines were

Copyright 2007 John Wiley & Sons, Ltd. J Gene Med 2007; 9: 797–805.DOI: 10.1002/jgm

Polyplexes Containing Masked Melittin 799

detectable by TNBS assay. The acylated melittin wasmixed with 1000 µl PLL-PDP (71 nmol PLL, 0.93 µmolPDP) diluted in 1 M guanidine hydrochloride, 0.5 M NaCl,20 mM HEPES, pH 8. Subsequent purification was carriedout analogously to the PLL-Mel conjugate procedure asdescribed above but using pH 8 buffers.

Erythrocyte leakage assay

Human erythrocytes were isolated from fresh, citrate-treated blood and washed in phosphate-buffered saline(PBS) by four centrifugation cycles, each at 800 g for10 min at 4 ◦C. The erythrocyte pellet was diluted 10-foldin 150 mM NaCl. Melittin, DMMAn-melittin and PLL-melittin conjugates were serially diluted in 90 µl buffer(HBS, pH 7.4) using a V-bottomed 96-well plate, resultingin melittin concentrations of 0.25–16 µM. For 100% lysis,control wells contained buffer with 1% Triton X-100. Avolume of 10 µl of erythrocyte suspension was added toeach well and the plates were incubated at 37 ◦C for30 min under constant shaking. After centrifugation at300 g for 10 min, 60 µl supernatant were analyzed forhemoglobin release at 405 nm using a microplate platereader (Spectrafluor Plus, Tecan Austria GmbH, Grodig,Austria).

Polyplex formation

Plasmid DNA encoding luciferase was condensed with PLLor PLL-melittin conjugates at different molar ratios of PLLepsilon-amino nitrogen to DNA phosphates (N/P ratio).

For instance, DNA/PLL polyplexes at N/P 2 wereprepared by mixing a solution of 2 µg of plasmid DNAin 50 µl HBG (Hepes buffered glucose; 20 mM Hepesplus 5% w/v glucose, pH 7.4) with a solution of 2.66 µgPLL in 50 µl HBG. For Tf-targeted polyplexes, polycationsPLL (N/P = 1.7)/PLL-Tf (N/P = 0.3) were applied, forTf-targeted and PEG-shielded polyplexes polycations PLL(N/P = 1.3)/PLL-Tf (N/P = 0.3)/PLL-PEG (N/P = 0.4)were applied. Polyplexes were rapidly mixed by pipettingand allowed to stand for 20 min at RT before use.

Measurement of particle size and zetapotential

The particle size of polyplexes was measured by dynamiclight scattering using a Malvern Zetasizer 3000 HS(Malvern Instruments, Worcestershire, UK). Polyplexeswere generated in HBG at DNA concentrations of20 µg/ml and subsequently diluted to 10 µg/ml. Forestimation of ζ -potential, polyplexes were diluted with10 mM NaCl to give a final DNA concentration of 2 µg/mland the zeta potential was measured as previouslydescribed [22].

Ethidium bromide (EtBr) displacementassay

Aliquots of PLL, PLL-Mel or PLL-DMMAn-Mel were addedstepwise to a DNA solution (20 µg/mL) in HBG containing400 ng/mL EtBr, and the decrease in fluorescencewas measured in a Varian Cary Eclipse fluorescencespectrophotometer (Varian, Mulgrave, Australia). EtBrfluorescence (exitation 510 nm, emission 590 nm) in theDNA solution prior to addition of polycation was set to100%.

Cell culture

Cell culture media, antibiotics and foetal calf serum(FCS) were purchased from Invitrogen GmbH (Karlsruhe,Germany). Cultured cells were grown at 37 ◦C in 5% CO2

humidified atmosphere. Neuro2A murine neuroblastomacells (ATCC CCL-131) were cultured in Dulbecco’smodified Eagle’s medium (1 g/L glucose) containing 10%FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and2 mM glutamine.

Luciferase reporter gene expression

Cells were plated in 96-well plates (TPP, Trasadingen,Switzerland) at a density of 104 cells (Neuro2A cells)per well 24 h prior to transfection. The polyplexes with200 ng of DNA (pCMVLuc) were added to the cellsin 100 µl fresh culture medium containing 100 U/mlpenicillin and 100 µg/ml streptomycin. For investigatingthe effect of endosomal acidification the transfectionmedium was supplemented with 200 nM of the inhibitorbafilomycin A1 (Alexis Biochemicals Corporation). Thetransfection medium was replaced after 3 h by 100 µl offresh culture medium. The gene expression was measured24 h after transfection. Detection of luciferase activitywas carried out as described recently [8]. Transfectionefficiency was evaluated as relative light units (RLU)per number of seeded cells (mean ± standard deviation(SD) of triplicates). Two nanograms of recombinantluciferase (Promega, Mannheim, Germany) correspondedto 107 RLU.

Metabolic activity

Cells were seeded in 96-well plates and treated after24 h with different amounts of conjugates ranging from2.4 to 24 µg/ml. After 3 h medium was replaced by100 µl of fresh culture medium. Metabolic activity of eachwell was determined using a methylthiazoletetrazolium(MTT)/thiazolyl blue assay after 24 h as follows: 10 µLof a 5 mg/mL solution of MTT in sterile PBS bufferwas added to each well. After incubation for 2 h at37 ◦C, the medium was removed, 100 µl of DMSOadded and samples were further incubated at 37 ◦C


800 M. Meyer et al.

for 30 min under constant shaking. Optical absorbancewas measured at 590 nm (reference wavelength 630 nm)using a microplate reader (Spectrafluor Plus), and cellviability was expressed as a percent relative to untreatedcontrol cells. Values of metabolic activity are presented asmeans ± SD of triplicates.

Cytotoxicity assay

Cells were seeded in 96-well plates and treated after24 h with different amounts of conjugates ranging fromfrom 2.4 to 14.8 µg/ml. Cytotoxicity was determined after30 min using the lactate dehydrogenase (LDH) releaseassay (Promega, CytoTox 96 nonradioactive cytotoxicityassay) according to the manufacturer’s instructions.

Results

Design and synthesis of thebioresponsive melittin conjugate

The aim of the current work was to create a bioresponsivePLL-based nonviral gene carrier. PLL belongs to theclass of synthetic amino acid polycations which can beionically complexed to nucleic acids. Due to the naturalamino acid backbone it is easily metabolized and thusshould possess a low long-term toxicity. Endosomolyticproperties and transfection activity however are very low.To overcome this limitation, we now grafted melittinonto the polycation. DMMAn modification of melittin wascarried out to mask the lytic activity in the extracellularenvironment (and thus reduce acute toxicity of thecarrier). Endosomal acidification is exploited to triggerthe lytic activity in the intracellular environment. Hencea relatively low cytotoxicity and high gene transferefficiency should characterize the bioresponsive genecarrier. The design is shown schematically in Scheme 1.

In order to synthesize the bioresponsive endoso-molytic gene carrier, DMMAn-Mel was conjugated toPDP-modified PLL by disulfide bond formation. DMMAn-Mel was produced using an excess of DMMAn. Excessof DMMAn in the reaction mixture which would causeundesired acylation of primary amines on PLL during thecoupling procedure was removed by filtration through aVivaspin ultrafiltration unit. Coupling with PLL-PDP wascarried out in the presence of 1 M guanidine hydrochlo-ride. Guanidine hydrochloride prevents aggregation ofthe negatively charged DMMAn-Mel and the polycationbefore coupling. Purification was performed by size exclu-sion chromatography with the column equilibrated at pH 8to avoid acidic cleavage of DMMAn. Resulting conjugateshad 7 to 10 molecules peptide per PLL.

Lytic activities of free peptides andconjugates

Erythrocytes were incubated with the free melittinpeptides and melittin-PLL conjugates in HBS, pH 7.4,

+

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O

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CIGA

CIGA

RKR QQKISWILPALLTTGVLKV

VLKV LTTG LPAL ISWI KRKR QQ+++

Scheme 1. Design of the bioresponsive melittin conjugate

for 30 min at 37 ◦C. The free peptide exposed high lyticactivity (>93% of haemoglobin release) at concentrationsof 4.5 µM and above which is consistent with previouslypublished work [7].

The lytic activity of the PLL-Mel conjugate was similar tofree melittin, suggesting that covalent attachment of PLLto melittin did not significantly affect the lytic activity.PLL-DMMAn-Mel conjugate showed relatively low lyticactivity at neutral pH (<20%). In contrast, lytic activitywas enhanced (80% haemoglobin release) after acidicpreincubation at pH 5 (Figure 1). At neutral pH, a long-term study (2.5 h preincubation of PLL-DMMAn-Mel at37 ◦C and pH 7.4) revealed that lytic activity at highestconcentration is still lower compared to the unmodifiedPLL-Mel conjugate (approx. 50% haemoglobin release,data not shown).

Biophysical characterization ofpolyplexes

In initial experiments DNA-binding properties wereevaluated using the EtBr displacement assay. PLL showedan efficient DNA condensation at N/P ratio of 1. In thecase of PLL-Mel and PLL-DMMAn-Mel conjugates higheramounts of polymer were necessary to achieve completereduction of EtBr fluorescence (approx. N/P 2) (Figure 2).



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Figure 1. Lytic activities of PLL conjugates. Washed humanerythrocytes were incubated with increasing conjugate concen-trations. The conjugate PLL-DMMAn-Mel was preincubated atpH 5 for 30 min at room temperature (open triangles) beforeperforming the assay. Erythrocyte lysis induced by melittin con-jugates at pH 7.4 is shown

Particle sizes and zeta potentials of polyplexes are listedin Table 1.

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Figure 2. DNA binding of PLL, PLL-Mel and PLL-DMMAn-Melconjugates. Complexes were formed in the presence of EtBr inHBG and inhibition of EtBr/DNA fluorescence was monitored.Values of relative fluorescence intensity are presented as means± SD of duplicates

Table 1. Particle size and zeta potential of polyplexes

PolyplexesParticle size

(nm)Zeta potential

(mV)

PLL 74 ± 8 22.6 ± 5.8PLL-Mel 133 ± 38 16.5 ± 1.1PLL-DMMAn-Mel 211 ± 29 17.2 ± 1.0PLL/15% Tf 95 ± 18 9.9 ± 1.5PLL-Mel/15% Tf 109 ± 29 13.3 ± 0.6PLL-DMMAn-Mel/15% Tf 233 ± 21 13.8 ± 0.7PLL/15% Tf/20% PEG 158 ± 79 3.7 ± 1.3PLL-Mel/15% Tf/20% PEG 144 ± 25 12.7 ± 0.9PLL-DMMAn-Mel/15% Tf/20% PEG 178 ± 38 3.7 ± 0.6

DNA polyplexes were prepared in HBG at an N/P ratio of 2 and aDNA concentration of 20 µg/ml.

Gene transfer efficiency of PLL-Mel andPLL-DMMAn-Mel polyplexes

In order to determine if conjugation of Mel or DMMAn-Mel improves gene transfer efficiency of PLL, polyplexesgenerated with PLL-Mel and PLL-DMMAn-Mel conjugateswere compared to free PLL polyplexes. Transfectionswere carried out at N/P ratios of 1 to 4. While PLLmediated only low gene transfer, PLL-Mel showed thehighest gene transfer activity at N/P 2. At higher N/Pratios the gene transfer activity of PLL-Mel decreased. Incontrast PLL-DMMAn-Mel mediated high gene transferactivity at all N/P ratios (Figure 3a). In parallel weperformed a cytotoxicity assay to detect the amountof LDH in the medium which was released after 3 hof transfection time (Figure 3b). While at N/P ratiosof 1 and 2 no LDH was detectable in the case of anypolyplex formulation, at higher N/P ratios the PLL andPLL-Mel but not PLL-DMMAn-Mel polyplexes inducedLDH release up to 15% and 25% LDH release (100%refers to completely lysed cells). A higher toxicity of thePLL and PLL-Mel polyplex formulations as compared toPLL-DMMAn-Mel also explains the decreasing luciferaseexpression at higher N/P ratios.

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Figure 3. Gene transfer with PLL, PLL-Mel or PLL-DMMAn-Melpolyplexes. Neuro2A cells were treated with pCMVLuc polyplexesat different N/P ratios (1 to 4). Luciferase gene expressionsat 24 h after transfection are shown in (a). White barsindicate PLL, black bars PLL-Mel and grey bars PLL-DMMAn-Mel.Luciferase activities are presented as means ± SD of triplicates.Corresponding LDH-release values are shown in (b)


802 M. Meyer et al.

To investigate the effect of endosomal acidificationon the gene transfer activity of the conjugates 200 nMbafilomycin A1, an inhibitor of vacuolar ATPase endosomepumps, was included in the transfection medium(Figure 4). As expected, the efficiency of standard PEIpolyplexes was about 7-fold reduced. Bafilomycin A1 didnot alter gene transfer of PLL polyplexes, but 3- and 8-foldenhanced gene transfer activity of the PLL-Mel conjugateat N/P ratios of 2 and 1, respectively. Gene transferby PLL-DMMAn-Mel was slightly (up to approx. 2-fold)reduced by the treatment (Figure 4).

Polyplexes containing transferrin (Tf) as targetingligand and polyethylene glycol (PEG) as shielding domainwere generated at an N/P ratio of 2 and comparedwith regard to reporter gene transfer. In all polyplexcompositions, linkage of Mel or DMMAn-Mel to PLLenhanced luciferase expression between 85-fold and morethan 1000-fold (Figure 5).

Toxicity of PLL, PLL-Mel andPLL-DMMAn-Mel conjugates

The toxicity of the conjugates is of major interest anddisplays a limiting issue for application of polyplexes. Toevaluate the differences in toxicity of PLL, PLL-Mel andPLL-DMMAn-Mel, relative metabolic activity of Neuro2Acells was determined after treatment with conjugates(Figure 6). PLL and PLL-Mel reduced the metabolicactivity of the cells to 15%, whereas, even for the highestconcentration of PLL-DMMAn, almost 50% of metabolicactivity remained. No significant differences were foundbetween PLL and PLL-Mel.

In contrast, differences in terms of cellular morphologywere detectable between all polymers already after1 h of incubation with polymers by transmission light

1E+03

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Figure 4. Effect of bafilomycin A1 on gene transfer. Luciferasegene expression after transfection of Neuro2A cells withPLL, PLL-Mel or PLL-DMMAn-Mel polyplexes in mediumsupplemented with 200 nM of the inhibitor bafilomycin A1.Luciferase activities are presented as means ± SD of triplicates

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65% PLL, 15% PLL-Tf, 20% PLL-PEG

Figure 5. Luciferase gene expression with different polyplexformulations. Luciferase gene expression after transfection ofNeuro2A cells with PLL, PLL-Mel or PLL-DMMAn-Mel polyplexes.Black bars indicate 100 (molar) % conjugate, white barsindicate 85% conjugate and 15% PLL-Tf, grey bars indicate 65%conjugate, 15% PLL-Tf and 20% PLL-PEG. Luciferase activitiesare presented as means ± SD of triplicates

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Figure 6. Metabolic activity of Neuro2A cells after conjugatetreatment. Neuro2A cells were treated with indicated concen-trations of PLL, PLL-Mel and PLL-DMMAn-Mel conjugates for3 h. Polymer-containing medium was replaced by fresh mediumand metabolic activity was determined after 24 h. Values ofmetabolic activity are presented as means ± SD of triplicates

microscopy (data not shown). Most significant changes incell morphology were found after addition of the PLL-Melconjugate. In the case of PLL-DMMAn-Mel no visible signsof toxicity were observed. Hence toxicity was furtherinvestigated by an LDH release assay. As illustrated inFigure 7, the amount of released LDH increased with theincrease in polymer concentration. The least LDH wasreleased by PLL-DMMAn-Mel, whereas PLL-Mel inducedthe most LDH release, which supports the microscopydata.

Discussion

The development of bioresponsive formulations (‘artificialviruses’) is a key issue in the field of nucleic aciddelivery [2,23–26]. Poor endosomal release and toxicity



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Figure 7. Polymer-induced lactate dehydrogenase (LDH) release.Neuro2A cells were treated with indicated concentrations of PLL,PLL-Mel and PLL-DMMAn-Mel conjugates. After 0.5 h incubationtime a LDH-release assay was performed. Values of LDH relativerelease are presented as means ± SD of triplicates

of the carrier limit the application of nonviral genetransfer systems. A variety of endosomolytic peptideshave been evaluated for the enhancement of nucleicacid delivery [2,5,8,27–29]. Among those for examplethe acidic peptide GALA [5] possesses a most favorablepH-specific lytic activity. The acidic influenza derivedpeptide INF7 shows pH-specific high activity also onnatural erythrocyte membranes and strongly enhancespolylysine (PLL)-mediated gene transfer [1]. The acidic,anionic nature of INF7 however also causes problems forformulation with DNA: PLL-INF7 conjugates similar tothose described previously [2] formed DNA complexeswhich were unstable and resulted in strong particleaggregation (data not shown). The cationic membrane-active peptide melittin does not display this problem;covalent melittin-polycation conjugates are well solubleand mediate strongly increased gene transfer activity[8,10,16].

Melittin displays similar highly efficient activity onbiological membranes as INF7; however, it is not pH-specific [1]. Lytic activity in the extracellular environmentis unfavorable as it mediates toxic side effects [7,21]. Toovercome this problem, Rozema et al. used an amine-reactive dialkylmaleic anhydride derivative to mask thelytic activity of melittin at neutral pH which was restoredafter acidic cleavage of the protecting groups [15]. Maleicanhydrides reversibly react with the lysine residues andthe N-terminal amino group of peptides and are removedagain at slightly acidic, endosomal pH [30].

Hence we built on this concept by now covalentlylinking a masked melittin peptide to a DNA-bindingpolycation. We linked dimethylmaleic anhydride maskedmelittin (DMMAn-Mel) to PLL, yielding the conjugatePLL-DMMAn-Mel (Scheme 1). For comparison we alsocoupled melittin peptide to PLL; such a conjugate has notbeen described previously.

Erythrocyte leakage assay (Figure 1) demonstrates thatPLL-DMMAn-Mel (in contrast to PLL-Mel) has relativelylow lytic activity at physiological pH over the wholeconcentration range. The lytic activity could be restoredalmost to the level of PLL-Mel after preincubation atendosomal pH. These results prove that dimethylmaleicanhydride shielding can be used to generate bioresponsiveconjugates with the desired lytic activity profile.

The lack of endosomolytic properties in the case of PLLcorrelates with its low gene transfer activity (Figure 3a).In contrast, both melittin conjugates greatly enhanced thegene transfer efficiency. A positive effect was observedwith a targeting ligand (transferrin) and a PEG shieldwithin the polyplex formulation (Figure 5). Such targetingand shielding domains have been found advantageous forin vivo gene delivery [31].

To investigate the effect of endosomal acidification onthe gene transfer activity, bafilomycin A1, an inhibitorof vacuolar ATPase endosome pumps, was includedin transfection experiments (Figure 4). The efficiencyof the gene carrier polyethylenimine (PEI) relies onthe ‘proton sponge’ effect upon acidification of theendosomal compartment [32]. Consistent with publishedliterature [33] inhibition of acidification by bafilomycinA1 strongly reduced the gene transfer activity. BafilomycinA1 did not alter gene transfer of PLL polyplexes whichlack proton sponge activity. Interestingly, bafilomycinenhanced gene transfer activity of the PLL-Mel conjugate.This can be explained by the higher lytic activity ofmelittin at neutral pH than at acidic pH [21]. Genetransfer by PLL-DMMAn-Mel was only slightly reducedby bafilomycin treatment. At first sight this incompleteinhibition seems to contradict the previous findings that30 min incubation of erythrocytes with masked melittindoes not result in lysis (Figure 1) and that bafilomycinA1 can inhibit membrane lytic activity of masked melittinwhen incubated with cells for 10 min [15]. However,we observed an increase in erythrocyte lytic activityof DMMAn-Mel at neutral pH after a long incubation(2.5 h at 37 ◦C) (see Results section). Altogether the PLL-DMMAn-Mel data are consistent with a slow unmaskingof melittin at neutral pH in the presence of bafilomycininhibitor. The lack of fast acidic pH-triggered unmaskingseems to counteract the enhancing effect of bafilomycinA1 as seen with non-masked PLL-Mel.

A slow unmasking of melittin at neutral pH overa longer time period should be sufficient in terms ofreduction of the acute toxicity, since polycationic genecarriers will be cleared from the blood circulation systemand extracellular space within relatively short periods[34,35]. It also might ensure enhanced transfectionefficiency upon intracellular trafficking of polyplexes intononacidic intracellular vesicles.

Apart from poor endosomal release also cytotoxicity ofgene carriers is one of the limiting barriers to systemicnonviral gene delivery. High lytic activity of polymersbefore reaching their target aggravates the problem. Toaddress this issue relative metabolic activity of conjugatetreated cells was determined via MTT assay (Figure 6). It


804 M. Meyer et al.

has to be emphasized that the transfection experimentswere performed at rather low polymer concentrations(2.4 µg/ml at N/P 2). Toxicity effects might be disguisedat low concentrations, and higher polymer amounts mightalso be necessary for in vivo delivery. Hence toxicity wasstudied over a broad concentration range. Notably, PLL-DMMAn-Mel was much better tolerated by cells resultingin higher metabolic activity than PLL and PLL-Mel.

To further evaluate the differences in toxicity we per-formed a lactate dehydrogenase (LDH) assay (Figure 7).The result confirmed that PLL-DMMAn-Mel is the con-jugate with the lowest acute toxicity. As expected,PLL-Mel mediated the highest LDH release. Remark-ably, PLL-DMMAn-Mel released less LDH than the non-endosomolytic PLL. In accordance with the observedvisible changes in cellular morphology PLL-Mel is the mosttoxic conjugate. It has to be mentioned that at higher poly-mer concentrations LDH release might also be enhancedbecause of cell necrosis and not only due to conjugate-induced membrane interaction and hole formation. Butalso at low concentrations (0 to 5 µg/ml) where most cellsare viable according to the metabolic activity assay differ-ences in LDH release are detectable. The observed resultsare in agreement with recent work on the interactionsof polycations with lipid bilayers and cell membranes[36,37]. PLL may induce lipid bilayer nanoscale hole for-mation and thus enhances membrane permeability dueto its regular positive charges. When the polycation isgrafted with DMMAn-Mel, parts of the regular polyca-tionic positive charges are masked and acute toxicity andmembrane-disruptive interactions are decreased. In con-trast, standard melittin which on its own can mediatemembrane disruption (as measured by LDH release; datanot shown) upon conjugation adds lytic activity to thepolymer. The data proves that DMMAn shielding of melit-tin not only avoids the undesired lytic activity in theextracellular environment, but also further reduces acutetoxicity of the core polycation PLL which is advantageousparticularly with regard to systemic in vivo delivery.

In summary, we have generated a bioresponsive genetransfer system based on the polycation PLL with triggeredendosomal lytic activity. Both melittin and DMMAn-Mel enhance gene transfer activity, but while melittinincreases toxicity, DMMAn-Mel grafting even lowersthe polycation cytotoxicity. These encouraging findingspresent hopefully one further step toward smart virus-like nucleic acid transfer systems [17,23,24]. Currently,investigations extending the findings to other polymericnucleic acid carriers are in progress.

Acknowledgements

We are grateful to Mrs. Olga Bruck, LMU Munich, for excellentassistance in preparing the manuscript. Wolfgang Rodl isacknowledged for his technical support. This work was fundedby the Deutsche Forschungsgemeinschaft SFB 486 ‘Nanoman’,Excellence Cluster ‘NIM’, and the EC project ‘GIANT’.

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a dimethylmaleic acid–melittin-polylysine conjugate with reduced toxicity, ph-triggered...

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