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Originally published In Press as doi:10.1074/jbc.M604876200 on May 9, 2007

J. Biol. Chem., Vol. 282, Issue 29, 21349-21360, July 20, 2007
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Crotamine Mediates Gene Delivery into Cells through the Binding to Heparan Sulfate Proteoglycans*

Fábio Dupart Nascimento{ddagger}1, Mirian A. F. Hayashi§2, Alexandre Kerkis, Vitor Oliveira||3, Eduardo B. Oliveira**, Gandhi Rádis-Baptista{ddagger}{ddagger}, Helena Bonciani Nader{ddagger}, Tetsuo Yamane§§, Ivarne Luis dos Santos Tersariol¶¶4, and Irina Kerkis||||5

From the {ddagger}Departamento de Bioquímica, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua 3 de Maio, 100, Ed. INFAR, CEP 04044-020, São Paulo, §Centro de Toxinologia Aplicada, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900, São Paulo, Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih Av. Brasil, 1085, CEP 01431-000, São Paulo, ||Laboratório de Neurociências, Universidade Cidade de São Paulo Rua Cesário Galeno, 448, CEP 03071-000, São Paulo, **Departamento de Bioquímica e Imunologia, Universidade de São Paulo Av. Bandeirantes, 3900, CEP 14049-900, Ribeirão Preto, {ddagger}{ddagger}Centro de Ciências Biológicas, Universidade Federal de Pernambuco Cidade Universitária, CEP 50732-970, Recife, Pernambuco, §§Instituto de Pesquisas Energéticas Nucleares, Universidade de São Paulo Av. Prof. Lineu Prestes, 2242, CEP 05508-000, São Paulo, ¶¶Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Av. Dr. Candido Xavier de Almeida Souza, 200, CEP 08780-210, Mogi das Cruzes, SP, and ||||Laboratório de Genética, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900, São Paulo, Brazil

Received for publication, May 22, 2006 , and in revised form, March 8, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell-penetrating peptides (CPPs)6 are molecules that display the distinguishing ability to enter eukaryotic cells through an energy-independent mechanism and to efficiently carry a number of biologically active and therapeutically relevant molecules into the cells. They are represented by multiple sequences of short and positively charged peptides rich in arginine and lysine residues that penetrate through usually impermeable cellular membranes and localize in the cytoplasm or in the nucleus of the cells or both (1–3). When used as carrier molecules, CPPs are usually covalently linked to the cargo or, alternatively, synthesized or expressed in tandem as a fusion protein with the peptide or protein cargos (4, 5). A list of cargo molecules delivered into various cells includes peptides, proteins, fragments of DNA, peptide-nucleic acid, small interfering RNA, liposomes, and magnetic nanoparticles (1, 2, 6, 7). However, particular approaches to attach them to the CPPs are always required. In addition, the usefulness of the CPPs as therapeutic agents has so far been hampered by the unspecific penetration of these carrier molecules into a vast range of cells (8, 9). Attempts have been made to confer the required specificity to some CPPs-cargo complexes (10).

The proposed mechanisms by which CPPs penetrate cells as independent molecules or carrying different cargos are still controversial. Several studies have suggested that CPPs translocate into cells via endocytosis (11), although non-endocytotic pathway cannot be excluded (12, 13). Some CPPs present electrostatic interaction with the extracellular matrix of the cell followed by endocytosis, whereas others appear to translocate across membranes by pore formation (11, 14). Lipid vesicles or membrane-associated heparan sulfate involvement in the uptake mechanisms have also been demonstrated (15).

Recently we described that crotamine, a myotoxin isolated from the venom of South American rattlesnake, is a CPP presenting both cytoplasmic and nuclear localization. At non-toxic concentrations crotamine penetrates into cells during G1/S period, binding to centrosomes and chromosomes. Accordingly, we have suggested the use of crotamine as a marker of centrioles, of cell cycle, and of actively proliferating (AP) cells (16). The specificity of crotamine to AP cells has also been demonstrated by conventional AP cells detection assay. The cells were incubated with 5-bromo-2'-deoxyuridine (5-BrdUrd) and with Cy3 fluorescent dye-conjugated crotamine (Cy3-crotamine) followed by immunostaining with anti-5-BrdUrd antibody, revealing superposition of the 5-BrdUrd and Cy3-crotamine labeling within the nucleus of the AP cells. Moreover, our previously published data suggested that crotamine penetration is a cell cycle-dependent event (16). Taken together, these findings indicate that crotamine is distinct from any other known natural CPPs.

Crotamine is a lysine-rich naturally occurring polypeptide whose structure is stabilized by three disulfide bonds (17, 18). The surface electrostatic potential of crotamine is characterized by a net charge of +10 (19, 20). The overall structural similarities between crotamine and other natural (8, 9, 12, 13) or synthetic, highly cationic poly-lysine-containing CPPs (21, 22) encouraged us to evaluate the ability of crotamine to function as a gene delivery system.

In the present work we investigated how crotamine and crotamine-plasmid DNA complexes gain access into a variety of AP cells both in vitro and in vivo. We monitored crotamine uptake and its intracellular trafficking, a sequential process involving interaction with cell surface heparan sulfate proteoglycans (HSPGs) followed by endocytosis and peptide accumulation within the lysosomal vesicles. Also, we demonstrated the effects of crotamine on the permeability of the endosomal/lysosomal membranes, which resulted in the leakage of these vesicles contents into the cell cytosol.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Lyso Tracker DND-99 and acridine orange (AO) were from Molecular Probes (Eugene, OR). Cell culture medium and supplements were from Invitrogen. Crotalus durissus terrificus venom was extracted from snakes maintained at the Faculdade de Medicina de Ribeirão Preto serpentarium, São Paulo University, and crotamine was obtained essentially as previously described (16). Wild-type Chinese hamster ovarian cells (CHO-K1) and their mutants deficient in all cellular glycosaminoglycans (GAGs) as a consequence of the removal of xylosyltransferase (CHO-745) were kindly donated by Dr. Jeffrey D. Esko (Glycobiology Research and Training Center, University of California, La Jolla, CA). The size-defined GAGs used were bovine lung heparin (10 kDa) (a gift from The Upjohn Co., bovine lung heparan sulfate (16 kDa) (a generous gift from Dr. P. Bianchini, Opocrin Research Laboratories, Modena, Italy), and dermatan sulfate (12 kDa), chondroitin sulfate (25 kDa), and chondroitinase ABC (from Seikagaku Kogyo Co., Tokyo, Japan); all were prepared by size exclusion column chromatography (23). Heparinase III (EC 4.2.2.8 [EC] ) and trypsin were purchased from Sigma. All other chemicals were from Sigma.

Peptide Binding to the Plasmid DNA—Electrophoresis mobility-shift DNA binding assay was performed by analyzing the migration of 2 or 4 µg of circular plasmid DNA (pEGFP-N1), pure or forming condensates with 10 µg of crotamine in agarose gel (1%, w/v) electrophoresis using Tris borate-EDTA buffer. The DNA alone or in complex with crotamine was stained with ethidium bromide in gel and was visualized by using a fluorescent image detection system (model FLA-2000, Fujifilm).

Dichroism Analysis—Circular dichroism (CD) experiments were carried out using a Jasco 810 spectropolarimeter (Jasco International Co. Ltd., Tokyo, Japan) coupled to a Peltier Jasco PFD-425S system for temperature control. Far UV-CD spectra were collected from 190–260 nm and averaged over 4–8 scans, with a 1-mm path length quartz cell. A 0.5-nm-step resolution, 50 nm/min speed, 8-s response time, and 1-nm bandwidth were used. The experiments were performed at 37 °C with 20 µM crotamine in 10 mM Tris-HCl, pH 7.4. The observed ellipticity was normalized to units of degrees·cm2·dmol-1. Base-line recordings were routinely made in the presence or absence of 20 µM GAGs. All data were obtained using three different solutions of the proteins. After base-line correction, the observed ellipticity [{theta}] (millidegrees) was converted to the molar mean residue ellipticity [{theta}] (deg·cm2·dmol-1) and used to correct the crotamine spectra.

Labeling of Crotamine with Fluorescent Dye—Fluorescent crotamine derivatives were prepared by using the FluorolinkTM Cy3-reactive dye (GE Healthcare) or the FluoReporter® FITC protein labeling kit (Molecular Probes, The Netherlands) following the instructions of the respective manufacturers. After labeling, the remaining free fluorescent dye was eliminated by using a Centricon spin column with a 3000-Da cut-off (Centricon 3 molecular weight cutoff concentrator, Amicon, Millipore Co.). The degree of labeling was estimated by absorbance measurements as indicated by the dye manufacturers and confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses (TofSpec-E, Micromass, UK).

Peptide-DNA Complex Formation—To produce crotamine-DNA complexes, the plasmidial vector pEGFP-N1 (Clontech, Mountain View, CA), which contains the green fluorescent protein (GFP) reporter gene, was used. Two and four µg of the plasmid DNA were incubated for 5 min with 10 µg of crotamine in 200 µl of 150 mM NaCl with occasional vortexing to form the peptide-DNA complexes.

Cell Lines and Culture Conditions—Mouse embryonic stem (mES) cell line USP 1 in passages 8–10 were grown onto a feeder layer of non-dividing, irradiated mouse primary embryonic fibroblasts under appropriate culture conditions (24). Human carcinoma cells HCT116 were cultured in McCoy's medium with 10% fetal calf serum and without antibiotics.

In Vitro and In Vivo Gene Transfection—All cells lines used in the in vitro studies were plated on 6 x 35-mm well plates using appropriate cell culture media. One day before transfection the cells were plated at a density calculated for each cell line reaching about 70% confluence in 24 h. On the day of transfection, the peptide-DNA complexes formed with 2 or 4 µg of DNA with 10 µg of crotamine in 0.2 ml of 150 mM NaCl were added dropwise to the cultured cells. In the control wells, 2 or 4 µg of plasmid DNA alone were added. Cells were incubated with the peptide-DNA complex for 6 and 24 h followed by a 24-h period in the absence of the complex, and then the cells were observed under confocal microscope. For the in vivo transfection assays, mice (n = 20) were injected intraperitoneally with 0.2 ml of complex solution containing 2 µg of DNA and 10 µg of crotamine or as a control group with 0.2 ml of solution containing plasmid DNA alone. Mice were sacrificed either 24 h or 30 days after intraperitoneal injection. The living cells from bone marrow (BM) and peritoneal liquid were isolated and observed directly under confocal microscope. Also, lung and liver tissues were dissected, and 6–10-µm cryostat sections were prepared using a Tissue-Tek embedding matrix (Sakura, Torrance, CA) and a cryo-microtome (Model CM 1100, Leica, Germany). Microscope slides were mounted in anti-fade and observed under confocal laser scanning microscope (LSM 510, Carl Zeiss, Jena, Germany).

Assay for Proliferative Activity of Cells—Proliferative activity of embryonic stem (ES) cells was analyzed by using FITC-conjugated anti-proliferating cells nuclear antigen antibody (Chemicon, Temecula, CA). About 106 cells were incubated with antibody for 30 min in an ice bath and washed in PBS plus 2% fetal bovine serum and 1 µM NaN3. Flow cytometry analysis was performed on fluorescence-activated cell sorter (BD Biosciences) using the CELL Quest program (BD Biosciences).

Binding of Crotamine to GAGs—A decrease in the fluorescence of crotamine (excitation at 280 nm and emission at 350 nm) after its binding to soluble GAGs was expected to occur. Thus, the intensity of this variation was taken as a measure of the binding and was used to determine the affinity of crotamine for heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate at 37 °C in 50 mM Tris-HCl buffer containing 100 mM NaCl, pH 7.4, unless otherwise specified. The fluorescence of the crotamine solutions was measured in a Hitachi-F2500 spectrofluorimeter with the excitation wavelength set at 280 nm (10 nm slit) and the emission scanned in the range of 300–400 nm (20 nm slit). The dependence of the relative fluorescence change, i.e. {Delta}F/Fo = (Fo - Fobs)/Fo, where Fo is the initial fluorescence value of the crotamine solution and Fobs is the observed fluorescence value after each GAG addition was analyzed by nonlinear least-squares data fitting by the binding equation (Equation 1) using the Grafit software,

Formula 1(Eq. 1)
where P is the total crotamine concentration, H represents the added GAG concentration, n is the stoichiometry, Kd is the dissociation constant, and {Delta}Fmax/Fo is the maximum relative fluorescence change.

The effect of the ionic strength on the dissociation constant was investigated by adjusting the buffer ionic strength with NaCl. The Kd value relates to the ionic strength according to the polyelectrolyte theory of Record et al. (25) by using Equation 2,

Formula 2(Eq. 2)
where KNI is the non-ionic dissociation constant at 1 M NaCl, Z is the number of ionic interactions, I is the ionic strength, and {Psi} is the fraction of bound counter-ion for each charged site on the GAG that is released upon binding to the crotamine.

Heparin-Sepharose Affinity Chromatography—The interaction of crotamine and heparin was assessed qualitatively by affinity chromatography. Crotamine and Cy3-crotamine (5 µM) were applied on heparin-Sepharose column (4 ml) previously equilibrated with 0.05 M sodium acetate buffer, pH 4.5. A linear NaCl gradient (0–1.0 M) was used to elute the bound material at a flow rate of 0.5 ml/min. The collected fractions were monitored at 280 nm for crotamine and by fluorescence detection for Cy3-crotamine. The fluorescence of Cy3 dye present in the Cy3-crotamine solutions was monitored by measuring the fluorescence at {lambda}em = 570 nm after excitation at {lambda}ex = 550 nm (10-nm slit) in a Hitachi F-2000 spectrofluorometer.

Cell Surface Labeling with Cy3-Crotamine—For binding and co-localization studies, CHO cells were kept at 37 °C in F-12 medium. CHO cells were grown on cover glasses, rinsed with PBS, and incubated with 0.5 µM Cy3-crotamine at 4 °C for 60 min in medium without serum. After washing cells were incubated for additional 60 min at 4 °C with FITC-labeled anti-heparan sulfate antibody (Seikagaku Co., Tokyo, Japan). The fluorescent signal of Cy3-crotamine and FITC-labeled anti-heparan sulfate antibody as well phase contrast micrographs of CHO cells were taken with confocal laser scanning microscope.

Also, for flow cytometry analysis CHO cells were harvested in PBS solution containing 5 mM EDTA and incubated with 0.5 µM Cy3-crotamine at 4 °C for 60 min (a condition that prevents endocytosis) followed by fluorescence-activated cell sorter analyses. Flow cytometry was conducted on a FACSCalibur flow cytometer with a 15-milliwatt, 570-nm, air-cooled argon ion laser and analyzed by the CELLQUEST software (BD Biosciences). Interaction of Cy3-crotamine with the CHO cell surface was determined as the percentage of cells emitting a fluorescent signal in FL2. The boundary between cells that stained positive and negative for Cy3-crotamine was determined according to the fluorescence distribution of positively stained, relative to unstained samples.

Cy3-Crotamine Binds Cell Surface HSPGs in CHO-K1 Cells—Cy3-crotamine (0.1–5.0 µM) dissolved in Tyrode's solution containing 0.1% bovine serum albumin was incubated at 37 °C for 120 min with CHO-K1 cells in the absence or in presence of 10 µM chondroitin sulfate or 10 µM heparin. Thereafter cells were washed with PBS and permeabilized with 0.5% Triton X-100 in PBS, and the amounts of Cy3-crotamine after cellular uptake were fluorometrically monitored. Also, CHO-K1 cells were preincubated with chondroitinase ABC, heparinase III, or trypsin. Thereafter, the cells were incubated with 2 µM Cy3-crotamine for 120 min at 37 °C, and the amount of Cy3-crotamine bound to the cell was fluorometrically determined.

Endocytosis of FITC-Crotamine by CHO Cells—The endocytic compartments of the CHO living cells were labeled with 1 nM Lyso Tracker DND-99 for 30 min at 37 °C. Thereafter, the cells were washed with F-12 medium and incubated with 0.5 µM FITC-crotamine. The fluorescent signal of Lyso-Tracker DND-99 and FITC-crotamine as well as the phase contrast micrographs were monitored at real time with a confocal laser scanning microscope.


Figure 1
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  		FIGURE 1.
Agarose gel shift assay and circular dichroism analysis of the peptide-DNA condensate. The peptide-DNA complex formation was examined by the electrophoretic mobility of the complex on agarose gel (A) or by CD spectra (B). A, the tests were performed by mixing 2 or 4 µg of the plasmid DNA (lanes 2–4 and 5–7, respectively) with 10 µg of the crotamine in the free form (lanes 2 and 5) or labeled with the Cy3 fluorescent dye (lanes 3 and 6) followed by electrophoresis through an agarose gel using Tris borate-EDTA buffer. Lanes 4 and 7 were loaded with the plasmid DNA alone. The DNA was then visualized after ethidium bromide staining. The lack or shift of migration was due to the neutralization of nucleic acid by the cationic peptide and/or formation of a large complex between the crotamine and the DNA. Lane 1 is a molecular weight marker (HindIII-digested {lambda}DNA). The DNA is the plasmidial vector pEGFP-N1 (4.7 kilobases) (Clontech, Palo Alto, CA). B, the CD spectra analysis of both crotamine and the plasmid DNA alone or in a mixture revealed that crotamine is able to complex with the plasmid DNA as suggested by the clear difference in the observed spectra of the mixture ({circ}) compared with the theoretically expected profile (•) of the curve obtained by the addition of individual spectra for DNA and crotamine alone.

 


Figure 2
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  		FIGURE 2.
In vitro gene transfer mediated by crotamine. Shown is the fluorescence-activated cell sorter analysis of proliferating cells nuclear antigen expression in mES cells (A) and negative control (B) using respective isotype. C–E, mouse ES cells treated with crotamine-plasmid DNA (pEGFP-N1) complex for 6 h showing a strong GFP fluorescent signal in ~20–30% of cells. The asterisk and arrows show non-transfected cells. C, fluorescent confocal microscopy (Fcm). D, differential interference contrast (DIC). E, overlay of DIC and Fcm. F–H, AP human carcinoma HCT116 cells, 24 h after treatment with the same complex. A strong GFP signal was observed in ~98% of cells. F, Fcm. G, DIC; H, DIC plus Fcm. C–E, bars = 100 µm. F–H, bars = 50 µm.

 
Intracellular Localization and Cellular Trafficking of Cy3-Crotamine—CHO-K1 cells were preincubated or not with inhibitors of endocytosis, namely 300 µM chloroquine, 20 µM chlorpromazine, 100 µM amiloride, and 10 mM beta-D-cyclodextrin at 37 °C for 60 min. Thereafter, the cells were incubated with 2 µM Cy3-crotamine for 120 min at 37 °C. After washing with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS, and the amounts of Cy3-crotamine after cellular uptake were fluorometrically monitored.

Time-lapse Microscopy—Time-lapse confocal fluorescence images taken under confocal laser scanning microscope (LSM 510, Carl Zeiss, Jena, Germany) were used for examining the localization of crotamine early in the endocytic process (5–30 min). For imaging, CHO-K1 cells were grown on cover glasses, and the living cells were labeled with 1 µM Cy3-crotamine and with 1 µM FITC-transferrin in F-12 medium without serum at 37 °C. Confocal images of Cy3-crotamine and FITC-transferrin fluorescence in living CHO-K1 cells were acquired at a 512 x 512-pixel resolution at a rate of 1 image every 5 min. The confocal optical image thickness was of about 1 µm.

Lysosomal Leakage Assays—To observe the lysosomotropic properties of crotamine, we have used the AO uptake and relocation methods as described previously (26). For imaging, CHO cells were grown on cover glasses, and the living cells were labeled with 5 µg/ml AO in F-12 medium without serum for 15 min at 37 °C. Thereafter, the cells were washed with F-12 medium and then exposed to 0.5–5 µM crotamine for different periods at 37 °C. The fluorescent signals of AO were taken with a confocal microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Crotamine Binding to Plasmid DNA—The peptide-DNA complexes were formed by mixing 10 µg of crotamine with 2 or 4 µg of the plasmid DNA. In this assay both crotamine in its free form or labeled with the Cy3-fluorescent dye were evaluated. Furthermore, it was observed that 2 µg of the plasmid DNA were sufficient to form a complex with 10 µg of crotamine, indicating a ratio of three DNA phosphates per molecule of crotamine. The peptide-DNA complex formation at the two aforementioned ratios was confirmed by the shift in the electrophoretic mobility of circular DNA in agarose gel (Fig. 1A). A lack or shift of the DNA migration due to the neutralization of nucleic acid negative charge by the cationic peptide and/or due to the formation of large complex between the crotamine and the DNA was observed for both circular and linearized DNA (data not shown).

Moreover, CD spectra analysis of both crotamine and plasmid DNA alone or in association revealed that crotamine is able to complex with the plasmid DNA as suggested by the clear difference observed in the obtained spectra of the mixture compared with the theoretically expected profile of the curve obtained by the addition of individual spectra for DNA and crotamine alone (Fig. 1B).

In Vitro Gene Transfer Mediated by Crotamine—We have previously shown that Cy3 fluorescent dye-conjugated crotamine selectively penetrates into the nuclei of AP cells (16). To verify if crotamine is able to selectively translocate the plasmid DNA into AP cells, mES cells were used in our study. These cells have an unlimited auto-renewal potential and are usually grown onto a feeder layer of mitotically inactivated mouse embryonic fibroblasts, which are non-dividing irradiated cells (27). Recently, we also showed that crotamine localization in non-dividing irradiated mouse embryonic fibroblasts was limited to the cytoplasm not being able to translocate into the nucleus of these cells (3). Thus, mES cell culture represents an interesting model that allows for the evaluation of the transfection efficiency of crotamine-plasmid DNA complexes in both AP and non-dividing cells. The AP nature of mES was confirmed by flow cytometry analysis using proliferating cells nuclear antigen antibody (Fig. 2, A and B).

For gene transfer crotamine-DNA complexes were diluted in salt solution and incubated with the cells for 6 and 24 h under appropriate culture conditions. A time interval of 6 h was used because in a cell population that is proliferating rapidly and asynchronously, 30–40% of the cells will be in S phase at any moment (28), which means that in ~24 h all AP cells would have progressed through mitosis. After this step, each culture medium was replaced by one without crotamine, and the cells were observed under a confocal microscope 24 h later. Control cultures were incubated with 2 or 4 µg of plasmid DNA alone. Mouse ES cells treated with crotamine-DNA complexes for 6 h showed a strong GFP fluorescent signal in ~20–30% of cells, indicating a selective pattern of crotamine penetration into the AP cells (Fig. 2, C–E). As expected, no signal was detected in either non-dividing mouse embryonic fibroblast cells (see the asterisk in Fig. 2, C–E) or in control culture cells treated only with the plasmid DNA (data not shown). Moreover, crotamine by itself was not able to induce cell proliferation in the cells used in this work (data not shown). Because the intracellular localization of the fluorescent signal could not be unambiguously ascertained within the islands of mES cells due to their juxtaposed growing, the AP human carcinoma HCT116 cells were also used in further experiments to overcome this limitation. Accordingly, after 24 h of incubation with crotamine-plasmid DNA complex, a strong GFP signal was observed in ~98% of the HCT116 cells (Fig. 2, F–H), demonstrating a high transfection efficiency of the crotamine-plasmid DNA complex. As expected, the GFP signal was mainly localized in the cytoplasm of transfected cells (Fig. 2, F and H). Furthermore, different cell types, such as murine fibroblasts 3T3 and human mesenchymal and melanoma cells were also efficiently transfected using the crotamine-DNA complexes (data not shown). As a rule, after 6 h of incubation with the complex, a fluorescent signal was observed only in part of the cells (~20–30%) for all cell lines studied, whereas after 24 h of incubation the majority of the cells (~98%) were transfected, suggesting a cell cycle-dependent mechanism of transfection.

In Vivo Gene Transfer Mediated by Crotamine—To verify if crotamine-plasmid DNA complexes would transfect in vivo, C57Bl/6 mice (n = 20) were injected intraperitoneally with a complex formed by mixing 10 µg of crotamine with 2 µg of the plasmid DNA, thus mimicking one of the conditions used in our in vitro studies. The animals were sacrificed 24 h after intraperitoneal injection of the complex, and the BM cells were immediately isolated and observed under a confocal microscope. A strong fluorescence signal was observed in ~10–20% of the BM cells (Fig. 3, A–F). We never observed transfection of all BM cells in vivo, probably because of the presence of a high number of non-dividing differentiated cells together with AP precursors (immature stem cells) (29). These precursor cells are likely the targets for the crotamine transfection. Curiously, we observed green fluorescence signal in about 20% of the BM cells obtained from animals sacrificed even 30 days after the injection of the crotamine-DNA complex (data not shown).


Figure 3
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  		FIGURE 3.
In vivo gene transfer after intraperitoneal injection of crotamine-plasmid DNA complexes into C57Bl/6 mice. A–C, strong GFP fluorescence signal visualized in ~20–30% of cells from the BM. D–F, higher magnification of cells showed in panels A–C showing GFP expression in the cytoplasm of BM cells. The arrowheads indicate untransfected cells. G–I, strong GFP signal revealed in the sinusoids emptying, where AP sinusoidal endothelial cells are usually located, and in the central vein areas of liver lobule. J–L, green fluorescence detected in highly regenerative bronchiolar epithelium of lung. M–O, no signal was observed in tissues isolated from control animal intraperitoneally injected with the plasmid DNA alone. M, BM. N, liver. O, lung. A, D, G, and J, Fcm; B, E, H, and K, DIC; C, F, I, L, M, and O, DIC + Fcm. A–C and G–O bars, 100 µm; D–F bars, 10 µm.

 


Figure 4
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  		FIGURE 4.
Biophysical properties of crotamine-heparin interaction. A, crotamine binds to the heparin-Sepharose column. Crotamine ({circ}) and Cy3-crotamine (•) bind the heparin-Sepharose column with the same affinity. AUF, arbitrary unit of fluorescence. B, effect of the ionic strength upon crotamine-heparin interaction. The effect of salt concentration upon crotamine-heparin interaction was investigated spectrofluorometrically at 0.1 M ({circ}), 0.2 M (•), 0.3 M ({square}), and 0.4 M ({blacksquare}) NaCl in 50 mM Tris-HCl pH 7.4, at 37 °C. The affinity of crotamine for heparin was quantitatively evaluated by direct titration monitored by fluorescence spectroscopy, in which the effect of discrete increments of the heparin concentration in the presence of 2 µM crotamine was recorded, showing an internal fluorescence of tryptophan residues of the crotamine decrease with heparin concentration increments. C, the effect of the ionic strength upon dissociation constant of crotamine-heparin interaction. The variation in the dissociation constant for crotamine binding over the ionic strength of the buffer is shown. D, interaction of crotamine with other glycosaminoglycan. The interaction of 1 µM crotamine with heparin ({blacksquare}), heparan sulfate (•), dermatan sulfate ({square}), and chondroitin sulfate ({circ}) were monitored spectrofluorometrically in 50 mM Tris-HCl buffer containing 100 mM NaCl pH 7.4 at 37 °C, showing that besides heparin only heparan sulfate showed a high affinity binding to crotamine. E, crotamine fluorescence emission spectra. The effect of heparin addition is observed by measurement of emission spectra, and no blue or red shifts were detected. Juxtaposed emission (at {lambda}ex = 280 nm) spectra are shown of 1 µM crotamine alone (•), 1 µM crotamine + 2 µM heparin ({square}), and 2 µM heparin alone ({circ}) in 50 mM Tris-HCl, pH 7.4, at 37 °C. F, effects of heparin on crotamine circular dichroism spectra. The 2 µM crotamine CD spectra were performed in the absence (•) or in the presence of 10 µM heparin ({circ}) in 10 mM Tris-HCl, pH 7.4, at 37 °C.

 
Besides BM cells, several other mouse tissues were dissected and fixed for cryostat section preparation. Fluorescence confocal microscopy analysis of 6–10-µm liver sections revealed a strong signal in the sinusoids and central vein areas of the liver lobule. A stronger signal was observed in some sinusoids emptying (Fig. 3, G–I), where AP precursors of sinusoidal endothelial cells are usually found (30). In the bronchioles of lung, a strong fluorescence was detected in highly regenerative bronchiolar epithelium (31), which is adjacent to smooth muscle that presented weak autofluorescent signal (Fig. 3, J–L). We also visualized a significant increase of fluorescent signal above the background level as compared with the control, peritoneal liquid and spleen cells, and in different areas of the muscle, heart, and brain tissues (data not shown). No fluorescent signal was observed in any of these tissues in control animals after intraperitoneal injection of the plasmid DNA alone (Fig. 3, M–O). Moreover, no adverse reactions were observed in mice after the intraperitoneal injection of the crotamine-plasmid DNA complexes.

Crotamine-GAGs Interaction—Next, to evaluate the binding of heparin molecules to the crotamine, a qualitative assay using a heparin-Sepharose affinity chromatography was employed. Both non-labeled and Cy3-crotamine were eluted from the heparin-Sepharose column at an ionic strength of 0.48 M (Fig. 4A). This result shows that the chemical coupling of Cy3-fluorophore to the crotamine did not disturb the interaction of crotamine with heparin-Sepharose resin and that this binding seems to be determined mainly by electrostatic interactions because of the relatively high ionic strength required to dissociate the complex. This raised the issue of specificity on the interaction of crotamine with heparin and how this interaction could affect crotamine structure. The affinity of crotamine for heparin was quantitatively evaluated by direct titration monitored by fluorescence spectroscopy as shown in Fig. 4B, in which the effect of discrete increments of the heparin concentration in the presence of 2 µM of crotamine was recorded. The internal fluorescence of tryptophan residues of the crotamine decreased steeply until the heparin concentration reached 1.0–1.5 µM, with a plateau at around 8.0 µM and above, indicating that these experimental data fit Equation 1. The variation in the dissociation constant for crotamine binding over the ionic strength of the buffer is shown in Fig. 4C. The panel shows the linear fit of the logarithm of the dissociation constants against the log of the ionic strength. According to the polyelectrolyte theory of Manning and Record (Equation 2), the y axis intercept is the log of the dissociation constant at 1 M salt and represents the strength of the non-ionic binding (KNI). The slope is related to the number of ionic interactions (Z) by the fraction of univalent counter ions released upon binding to the peptide, taken as 0.8 for heparin (32). For crotamine, the obtained slope was 1.6 ± 0.1, and the y axis intercept was 4.8 ± 0.1, yielding Z = 2.0 and KNI = 60 µM, respectively.


Figure 5
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  		FIGURE 5.
Interaction of crotamine with cell surface heparan sulfate proteoglycans. CHO-K1 (A and B) and CHO-745 (D and E) living cells were incubated with 0.5 µM Cy3-crotamine at 4 °C for 60 min. After washing, CHO-K1 cells were incubated for an additional 60 min at 4 °C with FITC-labeled anti-heparan sulfate antibody. The fluorescent signal of FITC-labeled anti-heparan sulfate (HS-FITC) antibody (G), Cy3-crotamine (H), and merged images (I) were taken with a confocal laser scanning microscope. Flow cytometry analysis is shown of CHO-K1 (C) and CHO-745 (F) cells incubated with 0.5 µM Cy3-crotamine at 4 °C for 60 min. A, B, and G–I, CHO-K1; D and E, CHO-745 cells. A, D, G, and H, Fcm; B, E, and I, DIC + Fcm; C and F, flow cytometry analysis. A, B, D, E, and G–I bars, 10 µm.

 
To investigate the specific polysaccharide sequence required for the interaction with crotamine, other polysulfated GAGs were tested. Dissociation constant (KD) values and the stoichiometry for crotamine-GAGs interaction are presented in Fig. 4D. Heparin binds crotamine with a dissociation constant of 0.73 ± 0.05 µM, whereas the values of KD for heparan sulfate were 1.4 ± 0.1 µM and for chondroitin sulfate and dermatan sulfate were 9 ± 1 and 12 ± 2 µM, respectively. That is, besides heparin, only heparan sulfate showed a high affinity binding to crotamine. All studied GAGs showed a stoichiometry value around 1.0, determined by the Hill plot as previously described (33), despite their KD values.

Fig. 4E shows the crotamine far UV-CD spectra obtained in the absence and presence of heparin. The crotamine CD spectra showed a positive band at 197 nm and a negative band at 207 nm, indicating a dominant antiparallel beta-sheet folding. The band in the region 221–231 is consistent with a Bb transition arising from Trp residues and/or with La (0->1) transition associated with the Tyr residues. Alternatively, this band can arise from the existing disulfide bonds in the crotamine structure between residues Cys-4—Cys-36, Cys-11—Cys-30, and Cys-18—Cys-37 (19, 20). The observed spectral changes caused by the presence of heparin are indicative of significant alteration in the secondary structure of crotamine. Similar spectral changes in a crotamine solution were induced by the presence of heparan sulfate or chondroitin sulfate (data not shown), indicating that different GAGs are capable of eliciting structural and probably functional changes in the protein.

The effect of 10 µM heparin on the emission spectra of a 2 µM crotamine solution is shown in Fig. 4F. Comparison of the spectra collected in the presence and absence of heparin indicated that there was an increase in the tryptophan fluorescence of the crotamine-heparin complex without any noticeable red or blue shift.

Crotamine Binding to Cell Surface Heparan Sulfate Proteoglycans—The aforementioned results demonstrated that heparin-like GAGs interact with crotamine in vitro. Next, the ability of Cy3-crotamine to bind cell surface HSPG was evaluated in ex vivo assay. Confocal fluorescence micrographs and flow cytometry analysis showed that crotamine internalization is a temperature-dependent process (Fig. 5). It is worthy mentioning that inhibition of endocytosis at low temperatures (4 °C) can be attributed not only to a blockage of the active process but also to the low fluidity of the lipid membrane at this temperature (34). Figs. 5, A and B, show the Cy3-crotamine accumulated on the cellular membranes of wild-type CHO-K1 cells at 4 °C. In contrast, we did not observed accumulation of Cy3-crotamine on the cell surface of CHO-745 cells (Figs. 5, D and E), which are almost completely devoid of GAGs. The binding of crotamine to cell surface HSPG was also quantitatively demonstrated by flow cytometry analyses (Fig. 5, C and F). Co-localization studies using FITC-labeled anti-heparan sulfate antibody confirmed that Cy3-crotamine interacts with extracellular HSPGs of CHO-K1 cells at 4 °C (Figs. 5, G–I).


Figure 6
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  		FIGURE 6.
Cy3-crotamine binds cell surface HSPG in CHO-K1 cells. A, Cy3-crotamine (0.1–5.0 µM) dissolved in Tyrode's solution containing 0.1% bovine serum albumin was incubated at 37 °C for 120 min with CHO-K1 cells in the absence ({blacksquare}) or presence of glycosaminoglycans: 20 µM chondroitin sulfate (•) or 20 µM heparin ({blacktriangleup}). Thereafter, the cells were washed with PBS, followed by permeabilization with 0.5% Triton X-100 in PBS. The fluorescence of Cy3-crotamine uptake by cells was fluorometrically monitored. AUF, arbitrary unit of fluorescence. B, CHO-K1 cells were incubated with 2 µM Cy3-crotamine at 37 °C for 120 min, and the amount of cell bound Cy3-crotamine was determined fluorometrically: control/untreated CHO cells (1), preincubated with chondroitinase ABC (2), heparinase (3), or trypsin (4). The values represent the mean value ± S.E., for n = 4.

 
Because the in vitro affinity of chondroitin sulfate for crotamine was only an order of magnitude lower than that of heparan sulfate, we decided to probe the specificity of Cy3-crotamine for GAGs in CHO-K1 cells. Fig. 6A shows that the interaction of Cy3-crotamine with CHO-K1 cells is saturable, displaying a hyperbolic relationship between Cy3-crotamine bound to cells as a function of the concentration of Cy3-crotamine. The observed dissociation constant of Cy3-crotamine with CHO-K1 cells was 0.8 ± 0.1 µM. This dissociation constant has the same value of dissociation constant determined for the interaction of crotamine with heparin; both values were 0.8 ± 0.1 µM (Fig. 4). Moreover, the fluorescence of Cy3-crotamine was significantly reduced when CHO-K1 cells were pre-incubated with 20 µM heparin (Fig. 6A) or when the cells were pretreated with heparinase (Fig. 6B). The preincubation of cells with 20 µM chondroitin sulfate (Fig. 6A) or the pretreatment with chondroitinase ABC (Fig. 6B) did not disturbed the Cy3-crotamine cell binding. Also, mild treatment of CHO-K1 cells with trypsin abolished the binding of Cy3-crotamine (Fig. 6B). Taken together, these data strongly support the conclusion that crotamine binding to the cell surface occurs via heparan sulfate proteoglycan.

Crotamine Uptake via Endocytosis—The inability of crotamine to penetrate into cells at low temperature strongly suggests that the internalization process involves endocytosis (16). To verify this hypothesis, the endocytic compartments of CHO cells were labeled with Lyso Tracker DND-99 at 37 °C for 30 min followed by the addition of FITC-crotamine into the cells. Confocal images of CHO-K1 unfixed cells obtained immediately after the dual labeling procedure showed FITC-crotamine localization within acidic vesicles, especially in lysosomes (Figs. 7A–C), suggesting that crotamine is taken up via endocytosis. Additionally, we observed that the FITC-crotamine uptake into endosomal compartment of CHO-K1 cells occurs within 6 min. As expected, we did not observe accumulation of FITC-crotamine inside CHO-745 cells (Figs. 7, D–F).

To better define the intracellular localization and cellular trafficking of crotamine, we studied the influence of classical endocytosis inhibitors on Cy3-crotamine uptake. The data presented in Table 1 show that the pretreatment of CHO-K1 cells with 300 µM chloroquine for 60 min prevents the endocytosis of Cy3-crotamine by about 92.3%. Note that chloroquine promotes the alkalinization of endosomal acidic vesicles (35). The functional role of clathrin-mediated endocytosis of Cy3-crotamine entry was assessed with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane (36). Pretreatment of the cells with 20 µM chlorpromazine inhibited the Cy3-crotamine uptake by 65%. On the other hand, the pre-treatment of the cells with either 10 mM beta-D-cyclodextrin, an inhibitor of lipid raft endocytosis (37), or 100 µM amiloride, an inhibitor of macropinocytosis (38), did not abolish the cellular uptake of Cy3-crotamine. To further illustrate the mechanisms by which crotamine gains access to these intracellular compartments, we used time-lapse confocal fluorescence images for examining the localization of crotamine at shorter times of endocytosis (5–30 min). As shown in Fig. 8, crotamine co-localizes with transferrin in the early phase of the endocytic process, corresponding to the initial 15 min after the addition of the fluorescently labeled proteins. These data indicate that Cy3-crotamine enters target cells by clathrin-mediated endocytosis followed by a fusion step within an acidic endosomal compartment.


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  		TABLE 1
Effect of endocytosis inhibitors on the Cy3-crotamine cellular uptake CHO-K1 cells were preincubated or not with the endocytosis inhibitors at 37 °C for 60 min. Thereafter, the cells were incubated with 2 µM Cy3-crotamine for 120 min at 37 °C. After washing with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS, and the cellular uptake of Cy3-crotamine was fluorometrically monitored. The fluorescence values of endocytosed Cy3-crotamine by CHO-K1 cells are represented by the mean ± S.E., n = 4.

 


Figure 7
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  		FIGURE 7.
Endocytosis of crotamine by CHO cells. The acidic vesicles of endocytic compartments of CHO-K1 (A–C) and CHO-745 (D–F) living cells were labeled with 1 nM Lyso Tracker DND-99 for 30 min at 37 °C. Thereafter the cells were washed with F-12 medium and incubated with 5 µM FITC-crotamine. The fluorescent signal of FITC-crotamine (A and D), LysoTracker DND-99 (B and E) as well as the DIC micrographs and merged images (C and F) were monitored at real time with confocal laser scanning microscope; these images were taken 6 min after FITC-crotamine incubation. Leakage of lysosomal/endosomal contents from CHO-K1 cells was monitored using vital AO in the absence (G–I) or in the presence (J–L) of 5 µM crotamine at 37 °C; these images were captured 30 min after FITC-crotamine incubation. Blue light excitation-induced AO red endosomal/lysosomal fluorescence (G and J), green cytoplasmic and nuclear fluorescence (H and K), and merged images (I and L) were also monitored in real time by confocal laser scanning microscopy. A–C and G–L, CHO-K1; D–F, CHO-745 cells. A, B, D, E, and G–L, Fcm; C and F, DIC + Fcm. A–L bars, 10 µm.

 


Figure 8
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  		FIGURE 8.
Endocytosis of fluorescent Cy3-crotamine and FITC-transferrin by CHO-K1 cells revealed by time-lapse confocal fluorescence microscopy. CHO-K1 cells were grown on cover glasses, and the living cells were labeled with 1 µM Cy3-crotamine and 1 µM FITC-transferrin in F-12 medium without serum at 37 °C. The elapsed time after the addition of the Cy3-crotamine and FITC-transferrin is indicated on each frame. Panels A–C follow the same field of CHO-K1 cells during Cy3-crotamine and FITC-transferrin incubation at 5 min (A), 15 min (B), and 30 min (C). The formation of endosomes containing both fluorescent Cy3-crotamine and FITC-transferrin is indicated by arrows. Notice the increase in intensity of fluorescence in endosomes over time.

 


Figure 9
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  		FIGURE 9.
Gene transfection into CHO cells. CHO cells were grown on cover glasses and incubated with 4 µg of plasmid DNA (pEGFP-N1) for gene transfection protocols in CHO-K1 (A) or in CHO-745 (B) cells. The dot squares show in detail the phase contrast of the cells image expressing the green fluorescence protein. Bars, 10 µm.

 
Crotamine Uptake Promotes Leakage of Endosomes/Lysosomes—The presence of multiple hydrophobic and proton-trapping basic amino acids in the structure of crotamine suggests that this molecule has lysosomotropic properties (39). As a consequence, because of the acidic lysosomal pH, the crotamine molecule could accumulate heavily inside the organelle inducing a disruption of the lysosomal membrane. The crotamine uptake-induced permeabilization of the endosomal/lysosomal membranes might, thus, result in the leakage of the vesicles contents to the cytosol, as previously described for other cationic peptides (40). We investigated this possibility by monitoring the fluorescent staining using the vital fluorogenic dye acridine orange. AO, a metachromatic fluorophore, accumulates mainly in the acidic vacuolar apparatus, preferentially in secondary lysosomes. When excited by blue light it shows red and green fluorescence at high (lysosomal) or low (nuclear and cytosolic) concentrations, respectively. Rupture of initially AO-loaded lysosomes may be monitored as an increase in cytoplasmic diffuse green fluorescence or a decrease in granular red fluorescence (26, 41, 42). The experiments were carried out by loading the CHO-K1 cells with the endosomal/lysosomal marker AO and then exposing them to crotamine. A marked increase in green fluorescence was observed upon incubation with crotamine, as seen by comparing the results shown in Figs. 7, H and K, indicating leakage of the AO from the lysosomes to the cytosol. Partial co-localization of red and green fluorescence was observed in CHO-K1 cells treated with crotamine (Fig. 7, J–L), strongly suggesting lysosomal rupture with release of the lysosomal AO to the cytosol. Note that normal CHO-K1 cells stained with AO showed a red fluorescence, which was restricted to the lysosomal vesicles (Fig. 7, G–I). The green fluorescence observed in these normal CHO-K1 cells was localized in the cytoplasmic and nuclear compartments (Fig. 7H) without any noticeable co-localization of the red and green fluorescence in these untreated cells (Fig. 7I), indicating the integrity of the lysosomal membrane. Taken together these results suggest that crotamine is capable of disrupting the lipid bilayer of lysosomal membrane. The leakage of the endocytic vesicles contents also promotes the crotamine spread to the cytoplasmic and nuclear compartments.

Role of Proteoglycans in the Cargo Delivery Mediated by Crotamine—Because proteoglycans and nucleic acids are potentially competitors of each other for electrostatic interactions with crotamine, we investigated the participation of HSPGs in the internalization process of crotamine-plasmid DNA complexes. Crotamine-mediated DNA delivery studies were performed in living CHO cells to avoid fixation artifacts (11). Crotamine was able to promote intracellular delivery of the pEGFP-N1 plasmid only into wild-type CHO-K1 cells, demonstrated by the intracellular expression of the associated reporter gene (Fig. 9A). On the other hand, there was almost no detectable GFP fluorescence in the GAG-deficient CHO-745 cell line (Fig. 9B), indicating that crotamine-heparan sulfate interaction is essential for the internalization of both free crotamine as well as crotamine-DNA cargo complex.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Snake venom is a very rich source of biologically powerful compounds, which act as toxins. The study of its composition allowed the discovery of a number of physiologically relevant compounds and in some cases the physiological pathway in which they are acting. Our data showed that crotamine, a myotoxin presenting CPPs-like properties, is capable of forming complexes with the plasmid DNA (Fig. 1), delivering it successfully into a wide range of cells in a living organism (Figs. 2 and 3).

The improvement of the in vitro transfection rate of plasmid DNAs after their attachment to natural peptides has been reported (43–45), but the resulting gene delivery systems generally lacked cell specificity. Here, we demonstrate that crotamine, which is a natural three-dimensional-structured peptide toxin, when employed as an in vitro gene delivery agent displays specificity to AP cells. It seems that this crotamine feature could be advantageous to mediate an efficient transfection of ES and other stem cells as compared with currently used procedures, such as electroporation, which causes the death of about 90% of the cells (46).

In addition, in the present work we demonstrate successful in vivo transfection in mice injected intraperitoneal with crotamine-DNA complexes. Moreover, we showed that the delivery of genetic material provided by the crotamine-pEGFP-N1 plasmid DNA complex was effective, since the GFP signal could be observed in BM cells by fluorescent confocal microscopy (Figs. 3, A and D). The proportion of BM cells displaying GFP fluorescence, about 10–20% of total, correlates well with the described ratio of proliferating cells present in the BM tissue (29). The presence of GFP signal was also investigated in the liver (Fig. 3G) and lung (Fig. 3J) of mice injected intraperitoneal with crotamine-pEGFP-N1 plasmid DNA complex, indicating that fluorescence was limited to cells from actively regenerating areas of the respective tissues (30, 31). Thus, the observed pattern of crotamine-mediated transfection in vivo is reminiscent of the previously described in vitro selective penetration of crotamine into AP cells (16).

To elucidate the mechanism of crotamine penetration, its interaction with the membrane-associated heparan sulfate was studied. Crotamine interaction with heparan sulfate and heparin was tested because among the GAGs they are particular in respect to their ability to bind a large number of different proteins and play a complex role at the cell surface, e.g. regulating a wide variety of biological processes, including hemostasis, inflammation, angiogenesis, growth factors, cell adhesion, and others (23). Furthermore, HSPGs are correlated to the cellular internalization of viruses, basic peptides, and polycation-nucleic acid complexes and, thus, possibly they have important implications for gene transfer and protein delivery to mammalian cells (49). In fact, the involvement of GAGs in uptake mechanisms has been shown for some CPPs (11, 15, 47, 48). Evaluating the effects of heparin and heparan sulfate chains on the binding of crotamine, we observed that crotamine interacts with heparin-Sepharose resin (Fig. 4A). This interaction seems quite specific, since other sulfated GAGs, namely chondroitin sulfate and dermatan sulfate, presented a much lower (about 10-fold) affinity to crotamine (Fig. 4D). As expected, the interaction between crotamine and heparin was disrupted by the addition of 0.5 M NaCl, showing that the binding of crotamine to heparin is governed mainly by electrostatic interactions (Figs. 4, B and C). Moreover, CD spectroscopy analysis confirmed the interaction of crotamine with heparin, since a consistent spectral change of the secondary structure could be observed (Fig. 4F). The results of this study demonstrate that crotamine internalization proceeds in a HSPG-dependent manner (Fig. 6). Table 1 and Fig. 8 data suggest that Cy3-crotamine enters CHO-K1 cells by clathrin-mediated endocytosis followed by a fusion step within an acidic endosomal compartment. It has been shown that Arf6 mediates syndecan recycling through endosomal compartments by a process controlled by syntenin PDZ domain-phosphatidylinositol diphosphate interaction. Syndecans that cannot recycle via this pathway are intracellularly trapped. This syntenin-mediated syndecan recycling pathway may regulate the surface availability of a number of cell adhesion and signaling molecules (50).

Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate and followed by escape from endosomes. The endosomal escape seems to be governed by the concentration of the endocytosed peptides and by the transmembrane pH gradient (inside acidic) across phospholipid bilayers (51). Once internalized, Cy3-crotamine was observed in the perinuclear space and in the cell nucleus of proliferative cells (16). Indeed, our data show that crotamine is capable of disrupting the lipid bilayer of acidic endosomal/lysosomal vesicles membrane (Fig. 7, J–L). At this point it is worthy mentioning that crotamine was shown to be nontoxic to the cells at the concentrations used (0.05–1.0 µM). Interestingly, we have identified that crotamine at concentrations above 5 µM was able to induce apoptosis by increasing lysosomal membrane permeabilization in transformed tumorigenic cell lines but not in normal AP cells (data not shown). It was recently demonstrated that transformation increases the susceptibility of cells to lysosomal death pathways (52, 53).

The presence of the crotamine in the nuclear compartment of the cells seem to be due to the leakage of the acidic endocytic vesicles contents, causing the spread of crotamine to the cytoplasmic and nuclear compartments, associated to the fact that crotamine has two putative nuclear localizations motifs, Crot2–18 and Crot27–39 (16).

HSPGs have also been detected in nuclear compartments (54). Syndecans are translocated to the nucleus in a time-dependent manner. The mitotic spindle shows more syndecans than that found in interphase cells (55). Glypican also has the putative nuclear localizations motifs. C6 cells entering the G1 phase acquire prominent foci of nuclear glypican immunoreactivity, suggesting that this nuclear localization may be related to the cell proliferation (56). Controlled nuclear entry of exogenous FGF-2 was observed around the G1 restriction point of the cell cycle and seemed to depend on cell proliferation (55). The internalization of FGF-2 is a consequence of syndecan-4 endocytosis (57). In a similar manner, crotamine seems to penetrate the proliferative cells in G1 phase, since the pericentriolar material associated with a pair of centrioles is strongly labeled by crotamine (16).

Pellegrin et al. (58) have shown that a cellular toxicity of CPPs is observed in AP cells and that this toxicity targets mainly proliferating cells and, to a lesser extent, confluent cells but neither quiescent nor differentiated cells. It was also observed that G1/S transition is an important event for cellular uptake of these peptides. Interestingly, the membrane potential varies during the cell cycle. A membrane hyperpolarization occurs in proliferating cell lines, particularly at the G1/S phase transition (58–60), due to the opening of K+ channels (59). Membrane hyperpolarization is a common phenomena observed in several proliferating cell lines (58–60). Henriques et al. (61) also showed that CPP uptake in HeLa cells was mainly dependent on the negative transmembrane potential across the bilayer, which suggests a physical mechanism governed by electrostatic interactions between CPPs (positively charged) and membranes (negatively charged).

HSPG is mainly synthesized by proliferative cells during G1 phase of the cell cycle (62, 63), and the membrane potential rises at the G1/S phase boundary and remains high throughout the M phases (60). Cellular hyperpolarization occurs if membrane potential rises to more negative values (64), i.e. when the cellular synthesis of HSPG is increased, the negative surface charges of membrane is also increased. The increased cellular synthesis of the highly anionic HSPG polymer during the G1/S phase can promote the opening of K+ channels, inducing hyperpolarization. Non-proliferating cells have a lower level of HSPG at cell surface than that of proliferative cells in G1/S phase (62, 63).

It has been shown that the ubiquitously expressed ClC-2 and ClC-1 chloride channels are mainly activated during cellular hyperpolarization (65, 66). ClC-type Cl- channels are responsible for endosomal Cl- conductance in many cell types, and so they are the major determinant of endosomal acidification (67), an active process controlled by a vacuolar H+ ATPase. Charge balance requires the accompaniment of inward H+ movement be accompanied by an influx of negative ions such as Cl-, which in most cells is the main ion responsible for shutting the interior-positive endosomal potential produced by active H+ entry (68). Endocytosis is mainly controlled by endosomal acidification, a process also involved in a variety of cellular processes, including vesicular fusion and budding, receptor and ligand sorting, and protein degradation (67, 69).

The results shown in Figs. 7, J–L, and Table 1 fit the general mechanism of gene delivery using non-viral vectors based on polyamine-DNA complexes, as postulated by the "proton sponge hypothesis" (70). It has been proposed that the high buffering capacity of polyamines containing titrate-able amines results in endosomal Cl- accumulation during acidification with presumed osmotic swelling and enhanced escape of polyamine-DNA complex. The observed escape of crotamine-DNA complex from endosomes (Figs. 7, J–L) and the effect of chloroquine, an inhibitor of endosomal acidification, in decreasing the cellular uptake of Cy3-crotamine (Table 1) are consistent with the proposed transgene delivery mechanism to the nucleus involving escape of the DNA-polyamine complex from endosomes, DNA/polymer dissociation, cytoplasmic DNA diffusion, and nuclear uptake (70).

The cellular uptake of crotamine is strongly related to the cell cycle. The uptake of crotamine into AP mES cells was demonstrated by flow cytometry analysis using anti-proliferating cells nuclear antigen antibody (Fig. 2, A and B).

Taken together our data suggest that crotamine is a potential candidate for establishing an effective DNA delivery into distinct AP cells both in vitro and in vivo. Previous publications (1, 2, 6, 8) have suggested the employment of CPPs as carriers of molecules into tissues of living organism. However, the main problem of CPPs to mediate the delivery of molecules in vivo generally is the unspecific spreading of the cargo through the whole body (71). In contrast to other natural CPPs and traditional carrier vectors used in gene delivery such as retroviruses and adenoviruses, which are mainly unspecific, crotamine displays the peculiar feature of delivering DNA vectors into AP cells of different tissues in a living organism. To the best of our knowledge this is the first report that proposes a potential application of a toxin as a molecular carrier.


   FOOTNOTES
 
* This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Med Discovery S.A., Ch. des Aulx 16, zip code 1228, Geneva, Switzerland. Back

2 Present address: Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio, 100, 04044-020, São Paulo, SP, Brazil. Back

3 Present address: Departamento de Biofísica, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio, 100, 04044-020, São Paulo, SP, Brazil. Back

4 To whom correspondence may be addressed: Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Prédio I, sala 1S-15, Av. Dr. Candido Xavier de Almeida Souza, 200, CEP 08780-210, Mogi das Cruzes, SP, Brazil. Fax: 55-11-4798-7102; E-mail: ivarne{at}umc.br. 5 To whom correspondence may be addressed: Laboratório de Genética, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP, 05503-900, Brazil. Tel.: 55-11-3726-7222 (ext. 2287); E-mail: ikerkis{at}butantan.gov.br.

6 The abbreviations used are: CPP, cell-penetrating peptide; HSPG, heparan sulfate proteoglycan; GFP, green fluorescent protein; ES cell, embryonic stem cell; mES cell, mouse ES cell; AP, actively proliferating; BM, bone marrow; GAG, glycosaminoglycan; AO, acridine orange; CHO, Chinese hamster ovary; Cy3-crotamine, Cy3-conjugated crotamine; DIC, differential interference contrast; Fcm, fluorescent confocal microscopy; CD, circular dichroism; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank the Center for Applied Toxinology (CAT/CEPID) for mass spectrometry analysis and also Dr. Katsuhiro Konno and Kátia Regina Brasil Melo for technical expertise.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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