Development and Characterization of New Cyclodextrin Polymer

Nov 24, 2008 - Institut de Chimie des Matériaux Paris Est, Syste`mes Polyme`res Complexes, Université Paris 12 Val de Marne, CNRS, 2-8 rue. Henri Du...
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Bioconjugate Chem. 2008, 19, 2311–2320

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ARTICLES Development and Characterization of New Cyclodextrin Polymer-Based DNA Delivery Systems Virginie Burckbuchler,† Ve´ronique Wintgens,† Christian Leborgne,‡ Sophie Lecomte,§ Nadine Leygue,§ Daniel Scherman,‡ Antoine Kichler,‡ and Catherine Amiel*,† Institut de Chimie des Mate´riaux Paris Est, Syste`mes Polyme`res Complexes, Universite´ Paris 12 Val de Marne, CNRS, 2-8 rue Henri Dunant, 94320 Thiais, France, FRE3087 CNRS-Ge´ne´thon, 1 rue de l’Internationale, BP 60, 91002 Evry Cedex, France, and Laboratoire de Dynamique, Interactions et Re´activite´, CNRS, 2-8 rue Henri Dunant, 94320 Thiais, France. Received February 22, 2008; Revised Manuscript Received September 18, 2008

In this study, we investigated whether a cyclodextrin polymer (polyβCD) complexed with cationic adamantyl derivatives (Ada) could be used as a vector for gene delivery. DNA compaction as a function of adamantyl/DNA phosphate ratio (A/P) by this new class of vector was demonstrated using surface enhanced Raman spectroscopy, ζ potential measurements, and DNA retardation assays. Transfection data highlight the relationship between in Vitro gene delivery efficiency and the combination of several physical properties of the polyβCD/Ada/DNA polyplexes, including cationic polar headgroup valency and chemical structure of the spacer arm of Ada connectors, the adamantyl/DNA phosphate ratio (A/P) of the polyβCD/Ada/DNA polyplexes, and the ionic strength of the medium. Finally, when associating the best formulation with a fusogenic peptide, we reached transfection levels which were of the same order as those obtained with DOTAP.

1. INTRODUCTION The delivery of DNA and other nucleic acid-based drugs such as siRNAs has a great potential in a wide variety of applications, including basic research and therapies for genetic and acquired diseases. However, the success of gene and siRNA therapies is largely dependent on the development of efficient delivery systems. One approach exploits the properties and tropisms of viruses. This is currently the most widely studied system, including in clinical trials (67% of the trials were done with viral vectors; for more details refer to the Web site http:// www.wiley.co.uk/genetherapy/clinical/). All other approaches, collectively termed “nonviral gene delivery systems” try to mimick the efficiency of viral vectors by artificial means (1). The most studied nonviral approach consists of using structurally well-defined synthetic compounds, among which a majority is cationic, such as polymers (2-4), lipids (5-7), and peptides (8-10). These molecules share several functions that are essential for transfection: besides condensing the DNA, they usually form positively charged particles which efficiently enter the cell after binding to negatively charged proteoglycans on the outer face of the membrane (11). Depending on the carrier, these cationic vectors can also facilitate the escape into the cytosol of the DNA complexes from the lumen of intracellular endolysosomal compartments. Despite the development of many compounds and a better understanding of the mechanism of transfection, the challenge of producing a nonviral vector with low toxicity and high * Corresponding author. Telephone: + 33 1 49 78 12 19. Fax: + 33 1 49 78 12 08. E-mail address: [email protected]. † Universite´ Paris 12 Val de Marne. ‡ FRE3087 CNRS-Ge´ne´thon. § Laboratoire de Dynamique, Interactions et Re´activite´, CNRS.

transfection efficiency still remains. Indeed, polyethylenimines and cationic lipids, which are the most used cationic transfection agents, were shown to have measurable cytotoxicity in Vitro and in ViVo (12-14). Only a few attempts have been made to correlate the influence of the physicochemical properties of DNA complexes on transfection efficiency. The first studies were done with cationic lipid-DNA complexes (15-18). Structures of DNA complexes based on cationic polymers have also been studied (19-21), but without correlating the physicochemical characteristics of the DNA complexes with their capacity to deliver DNA (22, 23). In this context, our work aims at both developing a versatile delivery system on the one hand and identifying the physicochemical properties required for an efficient gene transfer on the other hand. We are developing an innovative system resulting from the association between a cyclodextrin polymer (polyβCD) which has found interest in the field of gene therapy (24-26) and amphiphilic cationic connectors (Figure 1A). This association is obtained through the formation of inclusion complexes. Cyclodextrins (CD) are cyclic water-soluble molecules of 6 (RCD), 7 (βCD), or 8 (γCD) R-D-glucose units with a coneshaped architecture describing a cavity with an apolar interior which can induce an inclusion complex with a hydrophobic moiety (27, 28). The hydrophobic group of the amphiphilic cationic connector forms an inclusion complex with the CD cavity of the polyβCD, and this results in a “polycation”. The ternary complex, which is being called a “polyplex”, is achieved via cooperative electrostatic interactions between the cationic charge of the connector and the negatively charged phosphate groups of DNA. This system should present several advantages: (i) the charge density of the vector is easily controlled by a simple addition of a connector to the vector (polyβCD/ connector), (ii) it has been shown that CD can lower the cell

10.1021/bc800070f CCC: $40.75  2008 American Chemical Society Published on Web 11/24/2008

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Figure 1. (A) Scheme of polyplex assembly between the cyclodextrin polymer (polyβCD), the connector (Ada), and DNA. (B) Chemical structure of the Ada connectors.

Figure 2. Synthesis scheme of the Ada3 and Ada4 connectors.

toxicity in transfection experiments (29) and increase transfection efficiency (30, 31), (iii) this vector can also be used for cell targeting using adamantane derivatives coupled to a specific compound recognized by cell receptors (32-34), and (iv) the polyplex characteristics and thus the transfection properties can be modified by changing the connectors. In the present study, we have synthesized three different amphiphilic connectors (Figure 1B), which have in common the adamantyl hydrophobic group, which is well-known to make strong inclusion complexes, especially with βCD cavities. Several methods were then used to obtain information about DNA compaction and complexation. DNA mobility shift assays and ζ potential measurements allow the determination of the adamantyl/DNA phosphate ratio required for DNA complexation and the surface charge of the polyplexes. We have earlier elaborated a useful tool to study DNA compaction using surface enhanced Raman spectroscopy (SERS) (35). We can follow DNA compaction by monitoring the SERS signal of adenyl residues because the SERS signal of a nucleic acid mainly corresponds to the interaction of adenyl residues with silver colloid (Ag)n+. DNA compaction involves electrostatic interactions between the phosphate groups of DNA and the ammonium

group of the vector. These intermolecular interactions will compete with silver adsorption of adenyl. In this study we also focused on the role of ionic strength in DNA complexation by using DNA retardation assays and small angle neutron scattering. Indeed, in cell culture media the ionic strength is about 170 mM (36), which probably influences DNA complexation. Finally, the in Vitro transfection efficiency of these new DNA vectors was evaluated.

2. MATERIALS AND METHODS 2.1. Synthesis of the Cyclodextrin Polymer. The poly-βcyclodextrin polymer (polyβCD) was synthesized by polycondensation of βCD (a gift from Roquette Company, France) and epichlorohydrin under strong alkaline conditions (37). A full description of the polyβCD synthesis and the polyβCD characterization has been published (37, 38). The polyβCD sample used in this study has an average molecular weight of Mw ) 160 000 g mol-1 with a βCD content of 59% w/w, and its polydispersity index is Mw/Mn ) 1.9.

Cyclodextrin Polymer-Based DNA Delivery Systems

2.2. Synthesis of Adamantanyl Connectors. Ada2 (2-(1adamantyl)ethyltrimethylammonium bromide) was synthesized according to the previously published procedure (35). The synthesis scheme of Ada3 and Ada4 connectors is described in Figure 2, and the experimental procedure was as follows: Ada3 (2-(1-Carbamoylmethyladamantane)ethyltrimethylammonium Chloride). 1-Adamantane acetic acid (7 g, 36 mmol, Aldrich, Saint-Quentin-Fallavier, France) dissolved in 20 mL of thionyl chloride (Aldrich, Saint-Quentin-Fallavier, France) was placed in a distilled apparatus. After 1 h of reflux, thionyl chloride was removed by distillation and the apparatus was kept under vacuum. 5.8 g of 2-(1-adamantyl)ethanoyl chloride (1) as a pale pink liquid was obtained by distillation (75% yield). Compound 1 was characterized by 1H NMR (CDCl3, 300 MHz) δ (ppm): 1.8 - 1.7 (m, 12H; Ada), 2.1 (s, 3H; Ada), 2.7 (s, 2H; -CH2-CO). 2-(1-Adamantyl)ethanoyl chloride (1) (2.12 g, 10 mM) was added dropwise to a solution of N,N′-dimethylethylenediamine (0.8 mL, 7 mmol, Aldrich, Saint-Quentin-Fallavier, France) in 15 mL of pyridine previously placed under argon at 0 °C. Then, the mixture was stirred overnight and protected from the light. The product was evaporated three times with acetone to remove pyridine. The obtained solid was dissolved in a solution of H2SO4 (10%, pH ) 2.3) and then extracted with ether. Sodium hydroxide was added to the aqueous layer at 0 °C until pH ) 10 and then extracted with ether. Before being dried over anhydrous MgSO4, 2 g of product (2) was obtained as a beige solid (75% yield). Compound 2 was characterized by 1H NMR (CDCl3, 300 MHz): δ (ppm): 1.6 (m, 12H; Ada), 1.9 (m, 5H; Ada and Ada-CH2-), 2.2 (s, 6H; -N(CH3)2), 2.4 (t, 2H; -N-CH2), 3.3 (q, 2H; -CH2-NH). Compound 2 (2 g, 76 mmol) was dissolved in 5 mL of anhydrous dichloromethane, and then 1 mL of iodomethane (Aldrich, Saint-Quentin-Fallavier, France) was added drop by drop. The mixture was kept under reflux for 7 h and the solution was cooled to room temperature. Ether was added before filtration to obtain 2.6 g of Ada3 (80% yield) as a white solid. Iodide counterion was exchanged by chloride counterion with Dowex (2 × 8-100) anion exchange resin (Aldrich, SaintQuentin-Fallavier, France). Ada3 was characterized by 1H NMR (CDCl3, 300 MHz) δ (ppm): 1.6-1.7 (m, 12H; Ada), 1.9 (m, 3H; Ada), 2.0 (s, 2H; Ada-CH2-), 2.4 (t, 2H; -N-CH2), 3.5 (s, 9H; -CH3), 3.8-3.9 (m, 2H; -CH2-CH2-N). Ada4 (2-(1-Carbamoylmethyladamantane)di(ethyl(diethylmethylammonium)) dichloride). A solution of 2-(1-adamantyl)ethanoyl chloride (1) (1 g, 4.7 mmol, Aldrich, Saint-Quentin-Fallavier, France) in 10 mL of anhydrous ether was added drop by drop under argon to a solution of N,N,N′,N′-tetraethyldiethylenetriamine (1.33 mL, 5.1 mmol, Aldrich, Saint-Quentin-Fallavier, France) in a mixture of 30 mL of anhydrous ether and 0.65 mL of triethylamine. After 2 h of reflux, the white solid formed was filtered to eliminate triethylammonium chloride. The solution was evaporated to give 1.85 g (80% yield) of compound 3 as a yellow oil. Compound 3 was characterized by 1H NMR (CDCl3, 300 MHz) δ (ppm): 1.1 (m, 12H; -CH3), 1.7 (m, 12H; Ada), 1.9 (m, 3H; Ada), 2.1 (s, 2H; Ada-CH2-), 2.6 (m, 12H; -N-CH2-CH3 and-CO-N-CH2-CH2-),3.4(m,4H,-CO-N-CH2CH2-). To a solution of 1.5 g (3.8 mmol) of compound 3 in 50 mL of anhydrous dichloromethane was added drop by drop 1 mL of iodomethane. The mixture was kept under reflux for 2 h, and the solution was cooled to room temperature. The solution was filtered, and the solid product was washed with ether to give 1.8 g (70% yield) of Ada4 as a white solid. Iodide counterions were exchanged by chloride counterions with

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Dowex (2 × 8-100) anion exchange resin. Ada4 was characterized by 1H NMR (DMSO, 300 MHz) δ (ppm): 1.2 (m, 12H; -CH3), 1.6 (m, 12H; Ada), 1.9 (m, 3H; Ada), 2.1 (s, 2H; AdaCH2-), 3.0 (m, 6H; +N-CH3), 3.4 (m, 12H, -CO-N-CH2-CH2- and -CH2-CH3), 3.6 and 3.8 (m, 4H; -CH2-N+). 2.3. Polyplex Preparation. The vector composed of the polyβCD and the connector was generated by mixing a fixed amount of CD cavities of polyβCD [120 µmol · L-1 (10 equiv)] with various amounts of adamantyl connector ranging from 12 µmol · L-1 (1 equiv) to 120 µmol · L-1 (10 equiv). Polyplex formation was achieved by adding DNA plasmid solution in order to have a fixed 12 µmol · L-1 (1 equiv) of phosphate groups. All complexes were prepared in ultrapure water. PolyβCD/Ada/DNA polyplexes were prepared at various adamantyl/DNA phosphate ratios (A/P), which corresponds to the ratio between the number of adamantyl connectors and the DNA phosphate group number. 2.4. Physicochemical Characterization. 2.4.1. Fluorescence Measurements. The association constants (determined with a standard deviation of 10%) between the cyclodextrin derivatives and the connectors were measured with a SLM Aminco 8100 fluorimeter (Bioritech, Chamarande, France). A complete description of the method was published previously (39). A fluorescent probe, 4-amino-N-tert-butylphthalimide, able to make inclusion complexes with CDs, was used as a competitor (40) for Ada2, Ada3, and Ada4. The cyclodextrin derivatives used in these experiments are βCD, (2-hydroxypropyl)-β-CD (HPβCD) (Aldrich, Saint-Quentin-Fallavier, France), and the synthesized polyβCD. 2.4.2. DNA Retardation Assays. The polymeric vector binding to the DNA at different adamantyl/DNA phosphate ratios (A/P) was determined by using a 1% agarose gel electrophoresis (w/v) containing ethidium bromide in a TBE buffer (89 mM Tris/89 mM boric acid/2 mM EDTA, pH ) 8.3). Gels were run for 15 min at 100 V, and DNA was visualized using an UV illuminator. 2.4.3. ζ Potential. The ζ potential of the polyplexes were analyzed using a Zetasizer nano ZS (Malvern Instruments) at 25 °C. The measurements were performed in folded capillary cell (DTS 1060C, Malvern Instruments) with a 4 mW He-Ne laser at a wavelength of 633 nm. The ζ potential was automatically calculated from the electrophoretic mobility based on the Hemholtz-Smoluchowski relationship (41) for the polyplex polyβCD/Ada/DNA and based on the Hu¨ckel approximation for the vector polyβCD/Ada. 2.4.4. Surface Enhanced Raman Spectroscopy (SERS). The SERS experiments were carried out at 488 nm excitation of an argon ion laser on a Dilor XY Raman spectrophotometer (Horiba Jobin Yvon SAS, Longjumeau, France) equipped with a liquidnitrogen-cooled CCD camera. Silver colloids for SERS measurements were prepared according to the Creighton method (42) and display an absorption band centered at 398 nm measured on a Cary 3 UV-visible spectrometer (Varian, Les Ulis, France). Polyplex samples were obtained by mixing 90 µL of colloid with 10 µL of polyplex aqueous solution and 10 µL of a 1 mM aqueous solution of MgCl2. All SERS spectra were recorded after an incubation time of 20 min. The laser power was fixed at 40 mW at the sample, and the accumulation time was fixed at 180 s. 2.4.5. Small-Angle Neutron Scattering (SANS). The SANS experiments were carried out with the PACE spectrometer at the Laboratoire Le´on Brillouin (LLB), Saclay, France. The experimental scattering vector q (q ) (4π/λ) sin(θ/2)) range was 0.0032 < q (Å-1) < 0.12 and was covered by two sampleto-detector distances (3 m at the neutron wavelength of 6 Å-1 and 4.6 m at 13 Å-1). The wavelength dispersion ∆λ/λ of the

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velocity selector was 10%. The samples were kept in quartz cells (Hellma) with a path length of 2 mm for samples in D2O. The collected data were normalized for the detector efficiency using the isotropic scattering of a 1 mm thick calibration sample of H2O. The raw data were corrected for background from the solvent and sample cells. All the samples were prepared in D2O to maximize the contrast between the β-cyclodextrin polymer (FpolyβCD ) 2.29 × 1010 cm-2) and the solvent (FD2O ) 6.41 × 1010 cm-2). The scattering length densities of Ada2, Ada3, and Ada4 are 0.22 × 1010 cm-2, 0.46 × 1010 cm-2, and 0.23 × 1010 cm-2, respectively (without the counterions). In the SANS experiments, the DNA samples used are extracted from herring sperm (Sigma). Due to a lack of information regarding the relative composition in adenine, guanine, cytosine, and thymine, the scattering length density of the DNA sample could not been calculated (a theoretical value of 4.11 × 1010 cm-2 has been reported in the literature for the scattering length density of chicken erythrocyte nucleosomal DNA) (43). The concentration of each compound in the polyplex formulation is as follows: CpolyβCD ) 10-2 M (corresponding to 10 equiv of βCD cavities), CDNA ) 10-3 M (1 equiv of phosphate groups), CAda2 ) 5 × 10-3 M (5 equiv of adamantyl groups), and CAda3 ) 5 × 10-3 M (5 equiv of adamantyl groups). Because the scattered intensities of polyplex used under the concentration conditions of the other techniques are too weak to be detected, all sample concentrations are 10-fold higher in the SANS experiments. 2.5. Cell Culture and Transfection. In vitro cell transfection experiments have been performed using a plasmid DNA SMD2Luc∆ITR (7.6 kb) encoding a luciferase gene on the human hepatocarcinoma cell line HepG2 (American type Culture Collection, Rockville, MD) and the human transformed embryonal kidney cells HEK293 (American type Culture Collection, Rockville, MD). Cells were plated 24 h before transfection in 24-well plates at 37 °C in a 5% CO2/95% air incubator in a complete medium DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 1% (v/v) of the penicillin-streptomycin antibacterial solution (Invitrogen) and 10% (v/v) fetal bovine serum (Invitrogen). For transfection, cells were used at 50-80% confluency and all the tests were performed in triplicate. Culture media was removed and replaced by a mixture of 400 µL of serum free DMEM and 100 µL of polyplex aqueous solution at the desired adamantyl/DNA phosphate ratio (A/P) containing 2 µg of plasmid DNA per well. After 3 h, the transfection medium was removed and replaced with 1 mL of complete medium. Luciferase activity was measured 24-48 h posttransfection. The luciferase assay was performed as described (44). Luciferase background was subtracted from each value, and the transfection efficiency was expressed as total light units/ 10 s/well and is the means of the triplicates. The protein content of the transfected cells was measured by using the BioRad protein assay. Where indicated, the transfection experiments were done in the presence of the fusogenic peptide JTS-1 (Genepep), which has the following sequence: GLFEALLELLESLWELLLEA. The commercialized transfection agents PEI of 25 kDa (2) and the cationic lipid DOTAP (7) (N-(1-(2,3-dioleoyloxy)propyl)N,N,N-trimethylammonium chloride) were used without peptide. DOTAP was used at a ( charge ratio of 4 whereas, for B-PEI, we used an amine to phosphate ratio of 13. Notably, in preliminary experiments, we found that these ratios were optimal for the transfection of HepG2 and HEK293 cells. 2.6. Cell Viability Assay. Cytotoxicity was evaluated by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay (45). Briefly, one day after transfection, the cell culture medium was removed and replaced by serum-free DMEM containing 0.5 mg/mL MTT. After

Burckbuchler et al. Table 1. Logarithm of the Binding Constants (Determined with a SD of 10%) for Different Cyclodextrin Derivatives (βCD, HPβCD, and polyβCD) to the Adamantyl Connectors (Ada2, Ada3, and Ada4) and Their Variation with the Addition of NaCl log(KβCD) log(KHPβCD) + NaCl 150 mM log(KapppolyβCD) + NaCl 150 mM

Ada2

Ada3

Ada4

4.95 3.95 4.04 3.04 3.72

5.56 3.98

4.89 3.18 3.11 2.18

2.85

incubation at 37 °C for 3 h, the medium was removed and 200 µL of DMSO was added to each well to dissolve the formazan crystals produced from the reduction of MTT by viable cells. Absorbance was then measured at 570 nm. Untreated cells were used as control.

3. RESULTS AND DISCUSSION 3.1. Chemical Structure of the Connectors. The adamantyl connectors described in Figure 1B have been synthesized in order to evaluate the influence of the chemical structure on the transfection efficiency, in terms of the spacer and the charge valency of the polar headgroup. The three connectors have in common a hydrophobic adamantyl group, which is well-known to form inclusion complexes with CDs. The Ada2 connector has a spacer formed by an alkyl chain, and the polar headgroup is monocationic. On the other hand, Ada3 and Ada4 have a spacer composed by an amide function which is able to form hydrogen bonds with DNA (46-48). Furthermore, Ada3 has a monocationic polar headgroup whereas Ada4 is bicationic. 3.2. Fluorescence Measurements. Table 1 shows the logarithm of the binding constants, obtained by fluorescence measurements, of the three adamantyl connectors (Ada2, Ada3, and Ada4) for the cyclodextrin derivatives βCD, HPβCD, and polyβCD. These three cyclodextrin derivatives have different cavity accessibilities with an encumbering which ranks as follows: βCD < HPβCD < polyβCD. Adamantane derivatives show good affinities for βCD, as described in the literature (49). The logarithms of the binding constants are of the order of 5. HPβCDs have a higher steric hindrance than βCD due to their hydroxypropyl groups. This hindrance results in a decrease of the logarithm of the binding constants by one unit for Ada2 and by about 1.6-1.7 for Ada3 and Ada4 connectors. This corresponds to decreases from 20 to 30% of the binding energies. The measurements performed in a salt-rich medium in order to reduce the electrostatic repulsions of charges did not alter the binding efficiencies. Thus, the decrease of the binding constants is mainly due to a steric hindrance phenomenon. The lowest binding constants are obtained for polyβCD with Ada2, Ada3, and Ada4, as shown in Table 1. This is due to two complementary effects. On the one hand, the steric hindrance of the polyβCD, which is a branched cyclodextrin polymer, causes limited cavity accessibility. On the other hand, the polyelectrolyte behavior of the vector polyβCD/Ada generates unfavorable electrostatic repulsions between the connectors in solution and those which are already complexed in the CD cavity of the polyβCD. These electrostatic repulsions hinder complete complexation of free connectors. The Ada4 connector, with its two positive charges, is particularly sensitive to this effect, as shown by the low value of the logarithm of the binding constant (log(KapppolyβCD/Ada4) ) 2.18). This value is lower by one unity than the one obtained for the complexation of polyβCD with Ada2 (log(KapppolyβCD/ Ada2) ) 3.04). The increase of this later value with NaCl salt addition (log(KapppolyβCD/Ada2NaCl) ) 3.72) supports the explanation of unfavorable electrostatic repulsions (Table 1). 3.3. DNA Complexation and Compaction. 3.3.1. DNA Retardation Assay. We determined, in a first set of experiments,

Cyclodextrin Polymer-Based DNA Delivery Systems

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Figure 3. DNA retardation assay performed with the following DNA complexes: (A) Ada2 polyplexes, (B) Ada3 polyplexes, and (C) Ada4 polyplexes prepared at different adamantyl/DNA phosphate ratios (A/P).

Figure 4. ζ potential of the three systems Ada/DNA vector, polyβCD/Ada vector, and polyβCD/Ada/DNA polyplex, at different adamantyl equivalents (A eq) (1 A eq ) 12 µmol · L-1), as a function of the connector: (A) Ada2, (B) Ada3, and (C) Ada4. (D) Comparison of the polyplex behavior according to the type of Ada connector.

the ability of the different vectors to interact with plasmid DNA by performing a gel shift mobility assay which allows us to identify the amount of cationic vector required to inhibit the migration of the plasmid DNA during gel electrophoresis. Figure 3 shows the results of the DNA retardation assay using Ada2, Ada3, and Ada4 polyplexes prepared at an adamantyl/DNA phosphate ratio (A/P) ranging from 1 to 10. The first lane of each gel corresponds to noncomplexed plasmid DNA. Notably, the different bands represent three forms of the plasmid DNA. It is also important to mention that the connectors Ada2, Ada3, and Ada4 alone, used in the absence of polyβCD, are not able to compact DNA (data not shown). The resulting gels show that the different vectors altered the DNA migration, which underscores that polyβCD is an essential component of the vector system. For Ada2 and Ada3 polyplexes, full retardation occurs at about the same charge ratio (i.e., A/P between 2.5 and 5). Ada4 polyplexes inhibit DNA retardation slightly more efficiently than the two other systems, since an A/P ) 1 (equivalent to a ( ratio of 2) is sufficient due to its bicationic headgroup.

3.3.2. ζ Potential. In order to study more accurately the interactions of polyβCD/Ada with DNA, we performed ζ potential measurements of the polyplexes. Figure 4 reports the ζ potential as a function of the adamantyl equivalents (A eq) for the following different systems: connector/DNA, polyβCD/ connector, and polyβCD/connector/DNA. We observed the same behaviors for the three connectors Ada2 (Figure 4A), Ada3 (Figure 4B), and Ada4 (Figure 4C). First, a connector/DNA mixture generates a negative ζ potential, proving the inability of the connector alone to mediate DNA complexation. The addition of polyβCD permits us to obtain a positive ζ potential value, indicating a DNA complexation which is consistent with the gel electrophoresis results. Notably, the DNA association with polyβCD/connector induces lower ζ potentials than the polyβCD/connector alone. This surface charge decrease, which is expected, is due to the neutralization of cationic charges by the anionic charges of the DNA. Figure 4D compares the ζ potential variation of the three polyplexes as a function of the adamantyl equivalents (A eq). At low A eq, Ada2 and Ada3 polyplexes have negative ζ potential values, indicating the

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Figure 5. Influence of the nature of the connector on the SERS intensity of the polyplexes and on the percentage of free adenyl residues within the polyplexes.

presence of free DNA phosphate groups. Interestingly, a neutral surface charge of the polyplexes is obtained at an adamantyl equivalent of A/P ∼ 3.3 for Ada2, A/P ) 2.2 for Ada3, and of A/P ) 1 for Ada4 polyplexes. These results underline that the ability of Ada4 to complex DNA depends on the chemical structure of the connectors. Of note, the Ada3 vector offers better DNA complexation properties than Ada2, probably due to the amide function of its spacer, which can create hydrogen bonds with the DNA bases. Finally, the even more efficient DNA complexation properties of Ada4 may be based on the combination of two effects: hydrogen bonding as with Ada3 and the multivalency of the polar headgroup. 3.3.3. Surface Enhanced Raman Spectroscopy (SERS). The Raman signal intensity of an adsorbed molecule on or near special metal surfaces such as silver colloids can be enhanced by factors of up to 108 relative to the signal obtained for the

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same compound at the same concentration by classical Raman spectroscopy. This enhancement of diffusion permits the detection of molecules at very low concentration (10-6 M to 10-10 M) and then is particularly attractive for DNA studies. The SERS advantage is its ability to analyze the adenyl bases availability in nucleic acids. In fact, it has been proven that adenine is a very good SERS active molecule because free adenine has a specific affinity for silver colloids. Thus, the SERS signal of nucleic acids is mainly due to the interaction of these residues with the silver colloid. The spectral intensity of the observed SERS signal is a function of the availability of these residues. We have shown that the SERS method can be used to study the DNA compaction (35). The DNA compaction was analyzed in terms of the SERS signal decrease of adenyl residues by monitoring the intensity of the band at 1046 cm-1. This band is assigned to the vibrational motion of the C(6)NH2 site (35) and acts as a probe of the amount of adenyl adsorbed onto the silver colloids. Figure 5 shows the influence of the vector adamantyl/DNA phosphate ratio (A/P) on the adenyl residues accessibility of DNA at 1046 cm-1. The first point at the origin corresponds to the reference SERS signal intensity of DNA alone at 1046 cm-1. At low adamantyl/DNA phosphate ratio (A/P), the SERS intensity decreases strongly due to the formation of cooperative interactions between the cationic polar head of the connector and the anionic phosphate groups of DNA. Because of these interactions, which induce DNA reorganization, adenyl residues become more and more inaccessible to the silver colloids. The SERS intensity variation reveals an equivalent pattern for both polyβCD/Ada3 and polyβCD/Ada4 vectors whereas the polyβCD/ Ada2 vector shows a milder variation. This indicates again that the polyβCD/Ada2 vector has less efficient DNA compaction properties. The formation of efficiently compacted polyplexes occurs at the adamantyl/DNA phosphate ratio 2.5 < A/P < 5 for the Ada2 polyplexes and at 1 < A/P < 2.5 for both Ada3 and Ada4 polyplexes. The free adenine percentage within the

Figure 6. (A) Stability study of polyβCD/Ada2/DNA polyplexes at different adamantyl/DNA phosphate ratios (A/P ) 2.5, 5, 7.5) in the presence of various NaCl concentrations (CNaCl ) 25, 75, 100, and 150 mM). (B) Stability of polyβCD/Ada3 and Ada4/DNA polyplexes at different adamantyl/ DNA phosphate ratios (A/P ) 1, 2.5, 5, 7.5, 10) in water or in the presence of 150 mM of NaCl.

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Figure 7. (A) Ionic strength influence on the scattered intensities of polyβCD/Ada2/DNA (10/5/1) polyplexes and (B) comparison of the scattering behavior of the polyβCD alone in solution with the scattering behavior of polyβCD/Ada2/DNA (10/5/1) polyplexes in 75 mM and 100 mM salt concentration. (C) Ionic strength influence on the scattered intensities of polyβCD/Ada3/DNA (10/5/1) polyplexes and (D) comparison of the scattering behavior of the polyβCD alone in solution with the scattering behavior of polyβCD/Ada3/DNA (10/5/1) polyplexes in 75, 100, and 150 mM salt concentration.

polyplex was estimated at different adamantyl/DNA phosphate ratios (A/P) by the probe band at 1046 cm-1 and corresponds to the intensity ratio (Ipolyplex/IDNA)1046cm-1, where IDNA is the reference SERS intensity of DNA and Ipolyplex is the SERS intensity of the polyplex at the adamantyl/DNA phosphate ratio (A/P). Under the preparation conditions of the neutral polyplex (from the ζ potential data), Ada2 polyplexes have nearly 10% (A/P ) 3.3) of free adenyl residues, the Ada3 polyplex has 14% (A/P ) 2.2), and the Ada4 polyplex has 21% (A/P ) 1). The percentage of free adenyl residues decreases with an increase of the adamantyl/DNA phosphate ratio, which is an indication of the strength of DNA compaction. The Ada4 vector shows the highest free adenine percentage because the neutral polyplex was obtained at the lowest A/P ratio due to its bivalency. At highest adamantyl/DNA phosphate ratio,