Lower Rim Guanidinocalix[4]arenes: Macrocyclic Nonviral Vectors for

Apr 2, 2012 - Guanidinium groups were introduced through a spacer at the lower rim of calix[4]arenes in the cone conformation to give new potential no...
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Lower Rim Guanidinocalix[4]arenes: Macrocyclic Nonviral Vectors for Cell Transfection Valentina Bagnacani,†,§ Valentina Franceschi,‡,§ Laura Fantuzzi,† Alessandro Casnati,† Gaetano Donofrio,*,‡ Francesco Sansone,*,† and Rocco Ungaro† †

Dipartimento di Chimica Organica e Industriale, Università di Parma, Parco Area delle Scienze 17/A, I-43124, Parma, Italy Dipartimento di Salute Animale, Università degli Studi di Parma, Via del Taglio 6, I-43126 Parma, Italy

‡

S Supporting Information *

ABSTRACT: Guanidinium groups were introduced through a spacer at the lower rim of calix[4]arenes in the cone conformation to give new potential nonviral vectors for gene delivery. Several structural modifications were explored, such as the presence or absence of a macrocyclic scaffold, lipophilicity of the backbone, length of the spacer, and nature of the charged groups, in order to better understand the factors which affect the DNA condensation ability and transfection efficiency of these derivatives. The most interesting compound was a calix[4]arene unsubstituted at the upper rim and having four guanidinium groups linked at the lower rim through a three carbon atom spacer. This compound, when formulated with DOPE, showed low toxicity and transfection efficiency higher than the commercially available lipofectamine LTX in the treatment of human Rhabdomiosarcoma and Vero cells. Most of the investigated compounds showed a tendency to self-aggregate in pure water or in the presence of salts, as evidenced by NMR and AFM studies, and it was found that the ability to condense DNA plasmids in nanometric globules is a necessary but not sufficient condition for transfection. The superiority of macrocyclic vectors over linear Gemini-type analogues and of guanidinium compared to other ammonium head groups in determining the biological activity of the vectors was also ascertained.

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INTRODUCTION The design and synthesis of new nonviral vectors for cell transfection as an alternative to viruses is still an active area of research,1−3 due to the well-known problems related to the use of viruses, such as high toxicity, immunogenicity, and potential mutagenicity, and the limits experienced by the currently available synthetic gene delivery systems, such as poor efficiency and selectivity together with a not always negligible cytotoxicity. One of the most successful approaches in this field is the combination of a cationic lipid (cytofectin) with a neutral colipid,4 called “helper lipid”, because it allows a good balance between efficiency and toxicity. Since the first studies reported by Felgner,5 cationic lipids appear still as the most effective and widely exploited vectors for transfection protocols.1−3,6 Other systems, in particular based on polymers1,7−9 and dendrimers1,10−12 but also on nanoparticles1,13,14 and, more recently, macrocycles,15−18 have been tested and are used for these purposes. In the case of macrocyclic scaffolds, their conformational features can be exploited to obtain more preorganized vectors with possible positive effects on transfection efficiency. Cyclodextrins functionalized with cationic groups have been proposed as promising gene ligands and vectors,18−25 but before them, we reported several calixarene derivatives as the first examples of cationic lipids based on a macrocyclic structure. These compounds (e.g., I) were calix[n]arenes bearing guanidinium groups directly linked to the aromatic nuclei (upper rim)16,26 able to condense plasmid and linear DNA and to perform cell transfection in a way which © 2012 American Chemical Society

resulted strongly dependent on the macrocycle size, lipophilicity, and conformation. Unfortunately, they were characterized by low transfection efficiency and high cytotoxicity,16 especially at the vector concentration required for observing cell transfection (10−20 μM), even in the presence of the helper lipid DOPE27 (dioleoylphosphatidylethanolamine). With the aim of improving the transfection properties of this new type of nonviral vectors, we decided to shift the cationic head groups from the upper to the lower rim, privileging the calix[4]arene platform since the previous work with upper rim guanidinocalixarenes had shown that the calix[6]- and calix[8]arene derivatives were not able to compact DNA and give transfection.16 This manuscript reports the full details of the studies performed in our laboratories on lower rim guanidinocalix[4]arenes, which were partly described in a preliminary communication.28

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EXPERIMENTAL SECTION Procedures for DNA preparation, gel electrophoresis, melting curve determination, fluorescence and AFM experiments, cell culture, transient transfection, luciferase reporter, and MTT survival assays are reported in Supporting Information. Here, we report the procedure corresponding to the last step of the Received: December 23, 2011 Revised: February 16, 2012 Published: April 2, 2012 993

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Elemental analysis calcd (%) for C44H64N12O4Cl4 × 4H2O: C 54.66, H 6.67, N 17.38; found, C 54.51, H 6.49, N 17.28. 5,11,17,23-Tetra-n-hexyl-25,26,27,28-tetrakis(3guanidiniumpropoxy)calix[4]arene, Tetrachloride (5c). The pure compound was obtained as a white powder in quantitative yield. Hygroscopic. Mp >250 °C dec.; 1H NMR (300 MHz, CD3OD) δ = 7.75 (t, J = 5.4 Hz, 4H, NHCH2), 6.47 (s, 8H, ArH), 4.35 (d, J = 13.0 Hz, 4H, ArCH2Ar), 3.99 (t, J = 7.2 Hz, 8H, OCH2), 3.46−3.35 (m, 8H, OCH2CH2CH2), 3.13 (d, J = 13.0 Hz, 4H, ArCH2Ar), 2.32−2.15 (m, 16H, OCH2CH2 and ArCH2), 1.50−1.16 (m, 32H, ArCH2CH2CH2CH2CH2), 0.89 (t, J = 6.5 Hz, 12H, CH3); 13 C NMR (75 MHz, CD3OD) δ = 159.0, 155.4, 137.8, 135.8, 129.7, 73.5, 40.3, 36.6, 33.3, 33.1, 32.3, 31.1, 30.3, 24.2, 14.9. MS (ESI) m/z calcd for C68H112N12O4Cl4: 1157.8 [M-3H4Cl]+; found, 1158.0. Elemental analysis calcd (%) for C68H112N12O4Cl4 × 4H2O: C 59.37, H 8.79, N 12.22; found, C 59.14, H 8.52, N 12.03. 25,26,27,28-Tetrakis(6-guanidiniumhexyloxy)calix[4]arene, Tetrachloride (5d). The pure compound was obtained as a white powder in 87% yield. Mp >250 °C dec.; 1H NMR (300 MHz, D2O) δ = 6.89 (d, J = 7.6 Hz, 8H, ArH), 6.73 (t, J = 7.6 Hz, 4H, ArH), 4.51 (d, J = 13.1 Hz, 4H, ArCH2Ar), 4.00 (t, J = 6.8 Hz, 8H, OCH2), 3.33 (d, J = 13.1 Hz, 4H, ArCH2Ar), 3.22 (t, J = 6.3 Hz, 8H, CH2N), 2.06−1.95 (m, 8H, OCH2CH2), 1.71−1.57 (m, 8H, CH2CH2N), 1.57−1.41 (m, 16H, OCH2CH2CH2CH2); 13C NMR (75 MHz, CD3OD) δ = 158.9, 158.0, 136.5, 129.6, 123.4, 76.2, 42.9, 32.3, 31.6, 30.4, 28.2, 27.5. HRMS (ESI) m/z calcd for C56H88N12O4Cl4: 495.34420 [M-2H-4Cl]+; found, 495.34408. 49,50,51,52,53,54,55,56-Octakis(3-guanidiniumpropoxy)calix[8]arene, Octachloride (7). The crude was dissolved in water and purified by semipreparative RP-HPLC, with a C12 column using 30−60% gradient of CH3CN/TFA 100/0.05 v/v in H2O/TFA 100/0.05 v/v over 20 min. The fractions corresponding to the pure product were collected, and the solvent was evaporated under reduced pressure. The residue was solved (3×) in 10 mM HCl aqueous solution (5 mL), and each time, the solvent was removed under reduced pressure. The pure compound was obtained as a white powder in 27% yield. Mp >250 °C dec.; 1H NMR (300 MHz, D2O) δ = 6.91 (s, 24H, ArH), 4.04 (s, 16H, ArCH2Ar), 3.88 (t, J = 5.9 Hz, 16H, OCH2), 3.24 (t, J = 6.7 Hz, 16H, OCH2CH2CH2), 1.98 (quint, J = 6.2 Hz, 16H, OCH2CH2); 13 C NMR (75 MHz, CD3OD) δ = 158.9, 156.4, 135.8, 130.5, 125.8, 71.9, 40.1, 31.2, 30.8; HRMS (ESI) m/z calcd for C 88 H 128 N 24 O 8 Cl 8 : 411.25030 [M-4H+8Cl] 4+ ; found, 411.50098. 5-Guanidinium-25,26,27,28-Tetrakis(3-guanidiniumpropoxy)calix[4]arene, Pentachloride (11). The pure compound was obtained as a light yellow powder in quantitative yield. Hygroscopic. Mp 223−225 °C; 1H NMR (300 MHz, D2O) δ = 7.22−7.12 (m, 4H, ArH), 6.97 (t, 2H, J = 7.5 Hz, ArH), 6.54 (s, 3H, ArH), 6.37 (s, 2H, ArH), 4.40 (d, 4H, J = 13.4 Hz, ArCH2Ar), 4.18 (t, 4H, J = 7.1 Hz, OCH2), 3.99 (t, 2H, J = 6.9 Hz, OCH2), 3.96 (t, 2H, J = 6.9 Hz, OCH2), 3.48−3.28 (m, 12H, NHCH2 and ArCH2Ar), 2.35−2.16 (m, 8H, OCH2CH2); 13C NMR (75 MHz, CD3OD) δ = 159.0, 158.3, 157.7, 156.8, 156.1, 137.9, 137.6, 137.1, 135.4, 130.8, 130.4, 130.0, 129.3, 126.0, 124.3, 123.8, 74.1, 73.9, 73.5, 40.3, 40.2, 32.3, 31.2, 31.0; HRMS (ESI) m/z calcd for C45H68N15O4Cl5: 439.76665 [M-2H-4Cl]2+; found, 439.76602.

Chart 1. Upper rim guanidinocalix[4]arenes

synthesis of the ligands and their characterization. Synthetic procedures for all the new intermediates and their characterization are reported in Supporting Information. Melting points were determined on an electrothermal apparatus in capillaries sealed under nitrogen. 1H and 13C NMR spectra were recorded on Bruker AC300, AV300, and AV400 spectrometers. Partially deuterated solvents were used as internal standards; for 1H NMR spectra recorded in D2O at values higher than the room temperature, the correction of chemical shifts was performed using the expression δ = 5.060− 0.0122 × T (°C) + (2.11 × 10−5) × T2 (°C) to determine the resonance frequency of water protons.29 MS-ESI spectra were recorded on a Waters single quadrupole instrument SQ detector. HRMS-ESI spectra were recorded on an LTQ Orbitrap XL instrument. Elemental analyses were performed using a Termoquest 1112 CHN instrument and are reported as percentages. TLC was performed on Merck 60 F254 silica gel and flash column chromatography on 230−400 mesh Merck 60 silica gel. General Procedure for the Synthesis of the Guanidino-Calixarenes and Guanidino-Gemini from the Corresponding Boc Protected Precursors. Concentrated HCl (10 equiv for each Boc group) was added dropwise to a solution of the protected guanidinocalixarenes or guanidinylated Gemini derivatives in 1,4-dioxane (0.1 mmol/10 mL). The reaction mixture was stirred for 24−72 h, and the solvent was removed under reduced pressure to obtain the pure product. 5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis(3guanidiniumpropoxy)calix[4]arene, Tetrachloride (5a). The pure compound was obtained as a white powder in quantitative yield. Hygroscopic. Mp >250 °C dec.; 1H NMR (300 MHz, D2O) δ = 7.02 (s, 8H, ArH), 4.33 (d, J = 12.3 Hz, 4H, ArCH2Ar), 3.99 (t, J = 7.7 Hz, 8H, OCH2), 3.38−3.29 (m, 12H, OCH2CH2CH2 and ArCH2Ar), 2.29−2.25 (m, 8H, OCH2CH2), 1.10 (s, 36H, tBu); 13C NMR (75 MHz, CD3OD) δ = 158.7, 154.4, 146.1, 135.0, 126.4, 73.4, 40.0, 34.8, 32.3, 32.0, 30.9. MS (ESI) m/z calcd for C60H96N12O4Cl4: 1045.7 [M-3H-4Cl]+; found, 1045.7. Elemental analysis calcd (%) for C60H96N12O4Cl4 × 4H2O: C 57.04, H 8.30, N 13.30; found, C 56.90, H 7.95, N 12.92. 25,26,27,28-Tetrakis(3-guanidiniumpropoxy)calix[4]arene, Tetrachloride (5b). The pure compound was obtained as a white powder in quantitative yield. Hygroscopic. Mp >250 °C dec.; 1H NMR (300 MHz, D2O) δ = 6.86−6.80 (m, 8H, ArH), 6.80−6.70 (m, 4H, ArH), 4.40 (d, J = 13.3 Hz, 4H, ArCH2Ar), 4.08 (t, J = 7.0 Hz, 8H, OCH2), 3.40−3.33 (m, 12H, OCH2CH2CH2 and ArCH2Ar), 2.25 (quint, J = 6.9 Hz, 8H, OCH2CH2); 13C NMR (75 MHz, CD3OD) δ = 158.7, 157.2, 136.0, 129.6, 123.6, 73.2, 40.0, 32.1, 30.9. MS (ESI) m/z calcd for C44H64N12O4Cl4: 843.6 [M-3H-4Cl]+; found, 843.6. 994

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Scheme 1. Synthesis of the Lower Rim Guanidinocalix[4]arenes 5a−da

a Reagents and conditions: (i) N-(3-bromopropyl)phthalimide or N-(6-bromohexyl)phthalimide, NaH, dry DMF, N2, rt; (ii) NH2NH2·H2O, abs EtOH, N2, reflux; (iii) N,N′-di-Boc-N″-triflylguanidine, CH2Cl2, N2, rt; (iv) HCl 37%, 1,4-dioxane, rt.

Bis[5-tert-butyl-2-(3-guanidiniumpropoxy)-3methylphenyl]methane, Dichloride (21a). The pure compound was obtained as a white powder in quantitative yield. Hygroscopic. Mp >250 °C dec; 1H NMR (300 MHz, D2O) δ = 7.21 (s, 2H, ArH), 6.93 (s, 2H, ArH), 4.01 (s, 2H, ArCH2Ar), 3.79 (bt, 4H, OCH2), 3.35 (t, J = 6.0 Hz, 4H, OCH2CH2CH2), 2.27 (s, 6H, ArCH3), 2.07−1.90 (m, 4H, OCH2CH2), 1.17 (s, 18H, tBu); 13C NMR (75 MHz, CD3OD) δ = 159.0, 154.6, 148.0, 134.4, 131.5, 127.6, 127.5, 71.0, 40.3, 35.3, 32.2, 31.2, 30.8, 17.2. HRMS (ESI) m/z calcd for C31H52N6O2Cl2: 539.40680 [M-H-2Cl]+; found, 539.40695. Bis{[2-(3-guanidiniumpropoxy)-3-methyl]phenyl}methane, Dichloride (21b). The pure compound was obtained as a white powder in quantitative yield. Hygroscopic. Mp >250 °C dec.; 1H NMR (300 MHz, D2O) δ = 7.17 (d, J = 7.4 Hz, 2H, ArH), 7.00 (t, J = 7.4 Hz, 2H, ArH), 6.88 (d, J = 7.4 Hz, 2H, ArH), 3.99 (s, 2H, ArCH2Ar), 3.82 (t, J = 6.2 Hz, 4H, OCH2), 3.33 (t, J = 6.7 Hz, 4H, OCH2CH2CH2), 2.29 (s, 6H, ArCH3), 2.02 (quint, J = 6.4 Hz, 4H, OCH2CH2); 13C NMR (75 MHz, CD3OD) δ = 159.0, 156.9, 135.3, 132.4, 130.9, 130.0, 125.6, 71.0, 68.4, 40.2, 31.1, 30.8, 21.1, 16.9. MS (ESI) m/z calcd for C23H36N6O22Cl2: 427.3 [M-H-2Cl]+; found, 427.5. Elemental analysis calcd (%) for C23H36N6O22Cl2 × 2H2O: C 51.59, H 7.53, N 15.69; found, C 51.72, H 7.66, N 15.58. Bis[2-(3-guanidiniumpropoxy)-5-hexyl-3-methylhenyl]methane, Dichloride (21c). The pure compound was obtained as a white powder in 76% yield. Mp > 250 °C dec.; 1H NMR (300 MHz, CD3OD) δ = 7.44 (t, J = 5.1 Hz, 2H, CH2NH), 6.86 (d, J = 1.9 Hz, 2H, ArH), 6.63 (d, J = 1.9 Hz,

2H, ArH), 3.98 (s, 2H, ArCH2Ar), 3.78 (t, J = 5.9 Hz, 4H, OCH2), 3.42−3.32 (m, 4H, OCH2CH2CH2), 2.43 (t, J = 7.3 Hz, 4H, ArCH2), 2.27 (s, 6H, ArCH3), 2.02 (quint, J = 6.3 Hz, 4H, OCH2CH2), 1.55−1.40 (m, 4H, ArCH2CH2), 1.32−1.16 (m, 12H, ArCH2CH2CH2CH2CH2), 0.86 (t, J = 6.5 Hz, 6H, CH2CH3); 13C NMR (75 MHz, CD3OD) δ = 159.0, 154.8, 139.9, 134.9, 132.0, 130.7, 129.9, 71.0, 40.4, 36.5, 33.2, 33.0, 31.0, 30.8, 30.1, 24.0, 16.9, 14.8. HRMS (ESI) m/z calcd for C35H60N6O2Cl2: 595.46940 [M-H-2Cl]+; found, 595.46979. 25,26,27,28-Tetrakis(3-aminopropoxy)calix[4]arene, Tetrahydrochloride (12). Calixarene 3b was dissolved in aqueous 2 M HCl (2 mL) and stirred for 15 min, then the water was evaporated under reduced pressure to obtain the tetra salt as a white solid. Mp >250 °C dec.; 1H NMR (300 MHz, D2O) δ = 6.88 (d, 8H, J = 7.3 Hz, ArH), 6.80−6.73 (m, 4H, ArH), 4.39 (d, J = 13.3 Hz, 4H, ArCH2Ar), 4.14 (t, J = 7.2 Hz, 8H, OCH2), 3.41 (d, J = 13.3 Hz, 4H, ArCH2Ar), 3.14 (t, J = 7.4 Hz, 8H, CH2N+), 2.26 (quint, J = 7.8 Hz, 8H, OCH2CH2); 13C NMR (75 MHz, CD3OD) δ = 157.2, 136.3, 129.9, 124.0, 73.4, 38.8, 32.3, 29.6. HRMS (ESI) m/z calcd for C40H56N4O4Cl4: 653.40613 [M-3H-4Cl]+; found, 653.40568. 25,26,27,28-Tetrakis[3-(trimethylammonium)propoxy]calix[4]arene, Tetrachloride (13). The tetraminocalix[4]arene 3b (0.26 g, 0.4 mmol) was dissolved in MeOH (8 mL), then KHCO3 (0.41 g, 4.1 mmol) and MeI (0.8 mL, 12.7 mmol) were added and the mixture stirred at room temperature. The reaction was followed by mass spectroscopy and stopped after 7 days. A mixture MeOH/ CH2Cl2 9/1 v/v (10 mL) was added, the insoluble inorganic 995

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Scheme 2. Synthesis of Derivative 11a

salts were filtered off, and the solvent removed under reduced pressure. The residue (0.53 g, 0.4 mmol) was dissolved in H2O (5 mL), and 5 mL of Cl− DOWEX11 resin were added and the mixture stirred for 30 min. After removal of the resin, pure compound 13 was obtained by evaporation of the solvent under reduced pressure as a white solid in 98% yield. Mp 220 °C dec.; 1H NMR (300 MHz, D2O, 323 K) δ = 6.84−6.79 (m, 12H, ArH), 4.38 (d, J = 13.6 Hz, 4H, ArCH2Ar), 4.24 (t, J = 6.8 Hz, 8H, OCH2), 3.46 (d, J = 13.6 Hz, 4H, ArCH2Ar), 3.42− 3.28 (m, 8H, OCH2CH2CH2), 3.14 (s, 36H, CH3), 2.40−2.24 (m, 8H, OCH2CH2); 13C NMR (100 MHz, CD3OD) δ = 155.0, 134.8, 128.4, 122.5, 70.6, 63.7, 52.5, 31.1, 23.7. HRMS (ESI) m/z calcd for C52H80N4O4Cl4: 206.15394 [M-4Cl]4+; found, 206.15393.

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RESULTS AND DISCUSSION Synthesis. In order to obtain potential gene vectors with a different degree of lipophilicity, the three calix[4]arene scaffolds 1a, 1b, and 1c30 were selected for the functionalization with guanidinium units at their lower rim. This functionalization was performed by alkylation of the OH groups with propylphthalimido chains, subsequent removal of the amino protecting groups, reaction with bis-Boc-triflylguanidine, and final deprotection from Boc groups by reaction with HCl in dioxane, which gave the guanidinocalix[4]arenes 5a−c (Scheme 1). Using derivative 5b as a reference, we also performed a series of structural modifications in order to dissect the factors affecting DNA condensation and cell transfection in this family of ligands. We thus synthesized compound 5d (Scheme 1) to evaluate the effects of an increased distance between the apolar cavity and the charged groups, and we prepared octamer 7 (Scheme 3 and Scheme SI1, Supporting Information) to assess the influence of the platform conformational features and the higher number of charged head groups. As further modification, an additional guanidinium moiety with respect to 5b was introduced at the upper rim, synthesizing compound 11 through the reaction sequence described in Scheme 2. The last variation was the introduction of other positively charged groups instead of the guanidinium by the synthesis of compounds 12 and 13 (Scheme 3) bearing primary and quaternary ammonium cations, respectively. They were both prepared from 3b, the former by treatment with 2 M HCl and the latter by reaction with methyl iodide and a relatively weak base (KHCO3).31 To evaluate the importance of the macrocyclic structure in relation to the transfection properties, we also synthesized the series of Gemini-type compounds 21a−c (Scheme 3). These derivatives were obtained starting from the corresponding 2,2′-dihydroxydiphenylmethane precursors (Scheme SI2, Supporting Information) following the same reaction sequence (Scheme SI3, Supporting Information) used for their calix[4]arene analogues. Solubility and Aggregation Properties. All of the compounds synthesized were water-soluble, although within different concentration limits. They revealed aggregation properties which depend on their structure and could, in some cases, be responsible for their different behavior shown in DNA condensation and cell transfection (vide inf ra). The macrocyclic derivatives 5a, 5b, 5d, 11, 12, and 13 and the Gemini 21a and 21b gave rise to 1H NMR spectra in D2O at rt with sharp signals. On the contrary, the spectra of 5c and the corresponding model 21c showed broad signals as a reasonable consequence of hydrophobic aggregation due to the lipophilic hexyl chains on the aromatic nuclei. The sharpening of the

a

Reagents and conditions: (i) HNO3, glacial CH3COOH, dry CH2Cl2, N2; (ii) NH2NH2·H2O, Pd/C, abs EtOH, reflux, N2; (iii) N,N′-di-BocN″-triflylguanidine, Et3N, CHCl3, N2, rt; (iv) HCl 37%, 1,4-dioxane, rt.

signals upon dilution (e.g., Figure SI1 (Supporting Information) for compound 21c) confirmed the occurrence of this phenomenon. The signals of 5 mM 5c even at 363 K (Figure SI2a, Supporting Information) remained still rather broad, even though they could be recognized and assigned. Quite different was the situation in CD3OD (Figure SI2b, Supporting Information) where all the signals of 5c appeared sharp, indicating that in this solvent the aggregates are disrupted. Surprisingly, although apparently less amphiphilic compared to the calix[4]arene derivatives due to its conformational mobility, calix[8]arene 7 gave 1H NMR spectra in D2O (Figure SI3, Supporting Information) in which the signal shape and width are strongly dependent on the concentration, changing from broad to sharp by dilution and suggesting also for this derivative a high propensity to aggregation. A deeper investigation in water solution under varied conditions revealed further differences on the behavior of the members of this family of guanidinylated calixarenes. In particular, compounds 5a, 5b, 5d, 11, 12, and 13, which showed no evidence of aggregation in pure D2O at mM concentration, indeed act quite differently in the presence of inorganic salts. The addition of MgCl2 (2 mM) and NaCl (10 mM), both components of the deposition buffer used in the AFM experiments with DNA (vide inf ra), to a 1 mM sample of 5a in D2O determined a drastic reduction of the peak intensity in the 1H NMR spectrum, although no precipitation was observed. This suggests the formation of water-soluble aggregates in equilibrium with monomeric 5a. Analogously, calixarene 5b and 5d aggregate in the presence of the salt as evidenced by a significant broadening of their NMR signals, even if for 5b (Figure SI4, Supporting Information) a higher concentration of the two salts (MgCl2 50 mM and NaCl 250 mM) was necessary to cause clear changes in the spectrum. A polarity decrease of the medium determines disaggregation as demonstrated by the progressive sharpening of the NMR 996

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Scheme 3. Structures of Octamer 7, Ammonium Containing Ligands 12 and 13, and Gemini-Type Ligands 21a−c

rim analogues.16 The Gemini analogues of 5a−c and the macrocycles containing ammonium instead of guanidinium head groups are not able to interact efficiently with DNA even at a ligand concentration of 200 μM (Figure SI7, Supporting Information). Determination of the plasmid DNA melting curves in the presence of the ligands and evaluation of their ability to displace ethidium bromide from its complexes with the nucleic acid gave further, qualitative indication that binding between the DNA and all our ligands was taking place (Figures SI8 and SI9, Supporting Information). Using AFM in tapping mode on air, we could visualize and observe different effects of our compounds on DNA folding. After incubation of the circular plasmid pEGFP-C1 DNA (0.5 nM) with the p-tert-butyl calixarene 5a (0.6 μM) (N/P = 0.5) in the deposition buffer (4 mM Hepes, 10 mM NaCl, and 2 mM MgCl2, pH 7.4), the plasmids detected on the mica were 50−60 nm globular species (Figure 1b) each consisting of a single plectoneme.35 The presence of 10% of ethanol (v/v) in the deposition buffer where DNA and ligand molecules were dissolved during the incubation time caused a partial relaxation of the nucleic acid condensates (Figure SI10a, Supporting Information). A comparable behavior related to the presence or the absence of alcohol in the buffer was observed for compound 5c, although the condensates generated by this ligand (2.5 μM, N/P = 2) appeared even much larger and was often constituted of more than one plasmid (Figure 1c). On the contrary, compound 5b at 1−2 μM concentration, having no alkyl chains on the aromatic nuclei, did not form tight DNA condensates in the deposition buffer. Compared to their relaxed state (Figure 1a), the single plectomenes, however, appeared much more constrained, which proves the interaction of 5b with plasmid DNA. The addition of alcohol determined the formation of single plectoneme condensates, some of them detectable in the globular form (Figure 1d), others with a toroid-like36 shape (Figure SI10b, Supporting Information). Compound 5d, although lacking alkyl chains at the upper rim as 5b, seemed able to give, at 1 μM concentration, small condensates also in the absence of ethanol (Figure 1e). For all four calix[4]arenes 5a−d, it was also possible to observe on the mica surface very small aggregates of few nanometers not containing nucleic acid molecules, which could therefore be ascribed to the macrocycle aggregates previously observed in the absence of DNA. On the whole, ligands 5a−d seem to generate DNA globular species, of proper size for transfection in the case of 5a, 5b, and 5d, thanks to their ability to self-assemble through hydrophobic interactions between the alkyl chains at the upper rim (5a and 5c) or the aromatic nuclei of the calixarene (5b and 5d).

signals upon addition of increasing volumes of EtOH to the ligand samples in D2O solution containing salts (e.g., see the case of 5d in Figure SI5, Supporting Information). The data qualitatively revealed that this tendency to the aggregation follows the order 5c > 5a ≈ 5d > 5b. The self-assembly shown even by compounds 5b and 5d, although less markedly for the former one, indicates that also the unfunctionalized aromatic rings of the calixarenes themselves can actively participate in the formation of aggregated species through hydrophobic interactions not excluding for both compounds the possibility of condensing DNA and transfection. This represents an uncommon observation because, while it is well known that amphiphilic calixarenes functionalized with hydrophobic alkyl chains at the upper or lower rim tend to aggregate in aqueous solution,32,33 those without such substituents34 at the upper rim and with charged groups at the lower rim resulted as monomers in water,32 although no investigation of their behavior in presence of salts was reported so far. Accordingly, compound 11 did not show self-association at all since the additional charged group at the upper rim perturbs the polarity of the whole molecule disrupting any amphiphilic character. The presence of ammonium units, instead of guanidinium, eliminates the tendency to aggregate for the macrocycles 12 and 13 at least up to the investigated concentration of 1 mM, also with the simultaneous presence of MgCl2 and NaCl. AFM experiments with 5b and 5d (Figure SI6, Supporting Information) evidenced the same differences of behavior in pure water and in solutions containing salts (10 mM NaCl and 2 mM MgCl2) observed by NMR. The small aggregates visible on the mica surface by using buffer made clear that the two macrocycles at 1−2 μM are still able to aggregate despite a concentration much lower than that of the NMR studies (μM vs mM), probably thanks to the much higher salt concentration compared to that of the calixarene ligand. DNA Binding. The compounds obtained were then studied with different techniques to verify their binding properties toward DNA. All of the experiments were performed using the pEGFP-C1 plasmid DNA (4731 bp), encoding for the enhanced green fluorescent protein and subsequently used for the cell transfection assays. The ability of compounds to bind this plasmid was initially assessed through gel electrophoresis. The electrophoretic data (Figure SI7, Supporting Information) revealed that the guanidinium containing macrocycles strongly affect DNA mobility already at a concentration of 50 μM (cationic over anionic charges ratio N/P between ∼1/1 and ∼2/1 depending on the number of positively charged units on the ligand). It is also significant to notice that the efficiency of the lower rim guanidinocalix[4]arenes is higher than that observed, always by EMSA, for the previously described upper 997

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Supporting Information) to verify if the interaction with DNA plasmids visualized by AFM involves the macrocycles either as monomers or, following a hierarchical process,15,34,37−39 as self-assembled aggregates. In the case of 5c, a blueshift of the emission band and a strong enhancement of the fluorescence intensity were observed upon addition of the guanidinocalixarene (2 μM) to a buffer solution of the dye (0.2 μM). Subsequent addition of pEGFP-C1 DNA (0.5 nM) caused only a slight redshift of the emission band maintaining the same intensity. This suggests that, as recently reported for an amphiphilic tetrammonium-tetraoctyloxycalix[4]arene,34 compound 5c, endowed with long hexyl chains at the upper rim, forms aggregates that trap Nile Red and binds to DNA as self-assembled species. This conclusion, however, could not be clearly reached with 5a and 5b which showed a different effect on the Nile Red spectrum respect to 5c. Addition of these compounds to the solution containing the dye did not substantially change the fluorescence band which underwent a shift and an intensity increase only when DNA was added. In both cases, Nile Red did not interact with the calixarene aggregates, which we know from AFM experiments to be present in solution in these conditions, but it seemed instead to be included in the calixarene−DNA complexes. Therefore, for 5a and 5b, both possibilities are open: (i) the interaction with plasmid through a hierarchical assembly, in analogy with 5c, or (ii) the binding to DNA as monomeric species because of the lower aggregate stability due to the lack of long alkyl chains at the upper rim. AFM images relative to mixtures of DNA with the pentaguanidinium compound 11 at 1 μM concentration showed expected marked changes in the folding of the plasmids which, however, were mainly involved in the formation of very large aggregates (200−300 nm) (Figure SI10c, Supporting Information). Large aggregates were formed also by the guanidinocalix[8]arene 7, but together with other smaller condensates (Figure SI10d, Supporting Information) comparable in size with those observed, for example, with 5a and which, similarly, were relaxed by the addition of ethanol to the deposition buffer. Calix[8]arene 7 showed this behavior, which is in agreement with its observed self-aggregation ability even if it lacks alkyl chains at the upper rim and possesses a high conformational mobility compared to the rigid and amphiphilic cone calix[4]arene 5a. As previously verified by EMSA, the AFM experiments confirmed the lower efficiency of the two ammonium containing ligands 12 and 13 in the binding to DNA and their inability to condense it even when modifying the concentration (from 10−6 to 5 × 10−6 M) and the pH of the sample solutions (5.9 and 7.4) (Figures SI10e,f, Supporting Information). The data collected relative to these two compounds reveal that the presence of positive charges in this type of vectors is not sufficient to achieve efficient DNA binding and condensation and that guanidinium cations play a special role in this respect. While no substantial differences were revealed by EMSA in the binding properties of Gemini-type derivatives 21a−c, different effects on DNA folding attributable to the substituents at the aromatic nuclei of these compounds were observed by AFM measurements (Figure SI13, Supporting Information) also considering a higher N/P ratio not explored in gel electrophoresis. However, what was detected could not be related with the transfection properties of these ligands mainly because of their toxicity (vide inf ra).

Figure 1. AFM images showing the effects induced on plasmid DNA by the guanidinylated ligands. All images were obtained with the microscope operating in tapping mode in air and with supercoiled pEGFP-C1 plasmid deposited onto mica at a concentration of 0.5 nM. (a) Plasmid alone. Plasmid incubated with (b) 0.6 μM 5a; (c) 2.5 μM 5c; (d) 1.8 μM 5b + 10 % EtOH; (e) 1 μM 5d; and (f) 2 μM 5b + 4 μM DOPE using the cell growth medium as deposition buffer (90% DMEM, 10% FBS, 2 mM L-glutamine,100 IU/mL penicillin, and 10 μg/mL streptomycin). Each image represents a 2 × 2 μm scan.

Because of the particular efficiency in transfection characterizing the para-H lower rim guanidinocalix[4]arene 5b (vide inf ra), additional AFM experiments were performed upon incubation of the macrocycle with plasmid DNA in the cell growth medium (90% DMEM, 10% FBS, 2 mM L-glutamine, and 100 IU/mL penicillin and 10 μg/mL streptomycin) where DNA and a nonviral vector are indeed mixed for the treatment of cells. Having preliminarily verified that the medium allows the deposition of filaments on mica (Figure SI11a, Supporting Information), we proceeded with the analysis of samples coming from the incubation in such an environment of DNA and 5b with and without DOPE (ligand/DOPE 1:2), analogously to the used transfection experiment formulations. Condensates with proper shape and size for gene delivery were detected on the mica surface only for samples containing DOPE (Figure 1f and Figure SI11b, Supporting Information), similar to those previously observed with the same ligand in the absence of DOPE but upon the addition of 10% ethanol to the buffer solution (Figure 1d). This clearly evidenced the beneficial role of the helper lipid to obtain tight condensates and also how the experimental conditions given by the simpler deposition buffer represent a good model to collect information about DNA binding and the compacting abilities of these ligands in the more complex conditions of the transfection protocol. Fluorescence experiments with compounds 5a−c, at the same concentrations and with the same buffer of the AFM studies, were performed using Nile Red (Figure SI12, 998

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Transfection Properties. On the basis of the biophysical evidence of DNA binding and condensation, transfection experiments were performed on RD-4 human Rhabdomyosarcoma cells using plasmid pEGFP-C1 DNA (1 nM) encoding for the green fluorescent protein whose production in cells is easily detectable by fluorescence microscopy. This cell line was chosen because, beyond the medical relevance, it is easy to grow and difficult to transfect by traditional methods, especially if compared to other cell lines that are more widely used, such as HEK 293 cells. A first important observation is that no transfection occurred when either DOPE or ligands were used alone, also in the case of compounds which demonstrated DNA condensation ability in AFM experiments. On the contrary, the formulation calixarene/DOPE (1/2 molar ratio) especially at 10 μM (N/ P = 4) ligand concentration was effective for some of them and, in particular, for compound 5b which turned out to be a very efficient transfectant agent for RD-4 human Rhabdomyosarcoma cells. Remarkably, the amount of transfected cells for 5b (Figure 2), going from 35 to 50%, was higher than that achieved by the commercially available lipofectamine LTX (never more than 30% in our comparison experiments) and by our previously investigated upper rim tetraguanidinocalix[4]arenes I (less than 20%, Figure 2b). The presence of 10% of serum in the transfection mixture decreased to 11% the efficiency of 5b. Little transfection activity was observed for compounds 5a (3−4%) and 5c (6−7%). Quite rewarding was also the finding that the most active compound 5b has low cytotoxicity showing 70−75% of cell viability in MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays at 30 h from the treatment both by simple incubation (in the absence of DNA, Figure 3a) and in transfection conditions (in the presence of DNA, Figure 3b). On the contrary, cytotoxicity of 5a and particularly of 5c significantly increased going from their simple incubation with cells to transfection conditions (Figure 3). Rather surprisingly, also 5d, which showed a much lower efficiency in transfection experiments compared to 5b, overall appeared characterized by a very high cytotoxicity (Figure 3) which made irrelevant the determination of the transfection percentage. No significant differences in toxicity were revealed in the presence or in absence of DOPE for the four compounds. For a further elaboration of the transfection results, 5b and 5d were also compared by luciferase assays which supported the higher activity of 5b respect to both LTX (Figure SI14, Supporting Information) and 5d. Calix[8]arene 7 showed a maximum of 5% of transfection in the presence of adjuvant. The three derivatives 11, 12, and 13, all not showing self-aggregation propensity, were all unable to transfect Rhabdomyosarcoma cells. For compound 11, this result could be related to the formation of the very large aggregates of DNA observed on mica whereas for the other two to their inability to condense DNA (see the AFM studies above). The very low transfection found for Gemini compounds 21a and 21c is reasonably due also to their high toxicity. They were incubated at a double concentration with respect to the calixarene derivatives to have the same N/P ratio, and only 21b showed detectable transfection (ca. 6% at 20 μM). Since it is well known in the field of synthetic nonviral vectors that transfection efficiency may depend on the type of cell used, our lead compound 5b was tested in a different cell line setting using again LTX as reference. With Vero cells, again the calixarene based vector, together with DOPE, proved to be better than the lipofectamine, transfecting 30 and 20% of the

Figure 2. Transfection experiments performed with 1 nM pEGFP-C1 plasmid, guanidinocalixarene 5b/DOPE (1/2 molar ratio, 10/20 μM) formulation, and lipofectamine LTX to Rhabdomyosarcoma cells. (a) Fluorescence microscopy images (upper row) of the transfected cells as visualized thanks to the expression of the enhanced green fluorescent protein EGFP and phase contrast images (lower row) of the corresponding experiments. (b) In vitro transfection efficiency as percentage of transfected cells upon treatment with guanidino derivative/DOPE formulations and lipofectamine LTX.

treated cells, respectively.40 Comparable results between the two formulations were on the contrary obtained with AUBEK and BoMAK cells, whereas LTX was definitely more efficient in the case of hMSC and N2a cell lines. Experiments finalized at disclosing the uptake mechanism through which the complexes 5b/DOPE/DNA are internalized into the cells were also performed. Transfection of Rhabdomyosarcoma cells was carried out in presence of chemical compounds that inhibit different uptake pathways. On the basis of the preliminary results collected, macropinocytosis appeared as the most probable mechanism and caveolae-dependent endocytosis as a secondary pathway for the transport of these lipoplexes across the cell membrane. Wortmannin and amiloride, in fact known as inhibitors of macropinocytosis, and filipin, an inhibitor of caveolae-mediated uptake, significantly suppressed the transfection induced by 5b (Figure SI15, Supporting Information). In agreement with this assumption, we found that also monesine acts as an inhibitor of 999

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compound 5b for which the addition of DOPE, as observed by AFM, significantly helps in the condensation of the DNA plasmids.

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CONCLUSIONS In summary, attaching the guanidinium groups at the lower rim of calix[4]arenes disclosed the possibility to significantly enhance the cell transfection ability of the synthetic vectors based on this scaffold and reduce their toxicity to cells, if compared to the analogues I with the charged groups directly linked to the aromatic nuclei (upper rim). In particular, the macrocycle 5b was, in formulation with DOPE, even better than the widely used, commercially available lipofectamine LTX in the transfection of RD-4 human Rhabdomyosarcoma. The possibility to study and compare 5b with a series of analogues, in which structural modifications were introduced, evidenced that subtle variations in conformational freedom, distance of the charges from the cavity, nature of the cationic head groups and of the substituents decorating the apolar cavity, determine changes in the lipophilicity−hydrophilicity balance which in turn cause drastic changes in the biological properties also when relevant differences in DNA binding and condensation are not found by EMSA and AFM experiments. Moreover, we found evidence that the ability for these calixarene based vectors to self-assemble in water and condense DNA, as detected by AFM experiments,43 is a necessary but not sufficient condition for getting significant transfection activity. It is also interesting that, at least for lower rim derivatives in the cone conformation, the guanidinium is essential as a cationic group to observe transfection activity, and its replacement with other ammonium moieties is definitely detrimental, although some of these have been reported as effective when attached at the upper rim of calix[4]arene derivatives.34,44 The superior biological properties of these calixarene derivatives with respect to the linear Gemini-type compounds 21a−c indicate that a macrocyclic effect is operating and that the presence of a cavity and the preorganization of the charged groups make these derivatives promising systems to further develop interesting nonviral vectors.

Figure 3. Cell viability (Rhabdomyosarcoma cells) at 30 h from the treatment in MTT assays (a) after incubation with 10 μM ligands 5a− c and (b) in transfection conditions, with ligand alone (yellow bars), ligand/DOPE (10 μM/20 μM) formulation (orange bars), LTX (gray bar), DOPE alone (light blue bar), and without any treatment (blue bar).

transfection, and it is known that this compound blocks the pH induced degradation typical of the macropinocytosomes in which our complexes should reasonably be included. No effects on transfection were evidenced by inhibition of clathrin mediated endocytosis, which was indeed recently found out as one of the uptake mechanisms for cyclodextrin based vectors.41 We can thus put forward some hypotheses to explain, in particular, the superior performance found for the lower rim guanidinocalix[4]arene 5b as a nonviral vector compared to the upper rim guanidinocalixarenes I previously reported and shown to be less efficient and more toxic. We can rule out the fact that the different efficiencies could be linked to a possible different protonation degree between the lower and upper rim derivatives since we know that in all cases, the four guanidinium groups are fully protonated at physiological pH.42 A first reason could then be the flexibility of the chains linking the guanidinium groups to the macrocyclic scaffold of 5b which could favor the binding to DNA and cell membrane phospholipids. A further aspect seems related to the role played by DOPE on vector activity, which is in fact particularly relevant in the case of the lower rim guanidinocalixarenes and substantially unimportant for the upper rim derivatives I. The hydrophobic calixarene cavity of the former could allow an optimized interaction with the phospholipid helper exalting its functions and determining the formation of peculiar calixarene−DOPE supramolecular assemblies more suitable for transfection and endowed with much lower toxicity. This appears especially in the case of the most active para-H

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ASSOCIATED CONTENT

S Supporting Information *

Further synthetic schemes, experimental procedures, characterization, and 1H and 13C NMR spectra relative to all the new compounds; and pictures and graphics from gel electrophoresis analysis, AFM, fluorescence, melting curve determination, and cell transfection studies. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.D.); francesco. [email protected] (F.S.). Author Contributions §

These authors equally contributed to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Ministero dell′Istruzione, Università e Ricerca (MIUR, PRIN-project number 2008HZJW2L) and Fondazione Cassa di Risparmio di Parma which financially 1000

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biological properties of cyclodextrins bearing guanidinoalkylamino and aminoalkylamino groups on their primary side. Chem.Eur. J. 14, 4188−4200. (21) Diaz-Moscoso, A., Le Gourrierec, L., Gomez-Garcia, M., Benito, J. M., Balbuena, P., Ortega-Caballero, F., Guilloteau, N., Di Giorgio, C., Vierling, P., Defaye, J., Ortiz Mellet, C., and García Fernández, J. M. (2009) Polycationic amphiphilic cyclodextrins for gene delivery: synthesis and effect of structural modifications on plasmid DNA complex stability, cytotoxicity, and gene expression. Chem.Eur. J. 15, 12871−12888. (22) Diaz-Moscoso, A., Guilloteau, N., Bienvenu, C., Mendez-Ardoy, A., Blanco, J. L. J., Benito, J. M., Le Gourrierec, L., Di Giorgio, C., Vierling, P., Defaye, J., Ortiz Mellet, C., and García Fernández, J. M. (2011) Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery. Biomaterials 32, 7263−7273. (23) Bennevault-Celton, V., Urbach, A., Martin, O., Pichon, C., Guégan, P., and Midoux, P. (2011) Supramolecular assemblies of histidinylated α-cyclodextrin in the presence of DNA scaffold during CDplexes formation. Bioconjugate Chem. 22, 2404−2414. (24) Méndez-Ardoy, A., Urbiola, K., Aranda, C., Ortiz Mellet, C., García-Fernández, J. M., and Tros de Ilarduya, C. (2011) Polycationic amphiphilic cyclodextrin-based nanoparticles for therapeutic gene delivery. Nanomedicine 6, 1697−1707. (25) Méndez-Ardoy, A., Guilloteau, N., Di Giorgio, C., Vierling, P., Santoyo-González, F., Ortiz Mellet, C., and García Fernández, J. M. (2011) β-Cyclodextrin-based polycationic amphiphilic “click” clusters: effect of structural modifications in their DNA complexing and delivery properties. J. Org. Chem. 76, 5882−5894. (26) Dudic, M., Colombo, A., Sansone, F., Casnati, A., Donofrio, G., and Ungaro, R. (2004) A general synthesis of water soluble upper rim calix[n]arene guanidinium derivatives which bind to plasmid DNA. Tetrahedron 60, 11613−11618. (27) Farhood, H., Serbina, N., and Huang, L. (1995) The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim. Biophys. Acta, Biomembr. 1235, 289−295. (28) Bagnacani, V., Sansone, F., Donofrio, G., Baldini, L., Casnati, A., and Ungaro, R. (2008) Macrocyclic nonviral vectors: high cell transfection efficiency and low toxicity in a lower rim guanidinium calix[4]arene. Org. Lett. 10, 3953−3956. (29) Gottlieb, H. E., Kotlyar, V., and Nudelman, A. (1997) NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512−7515. (30) Shinkai, S., Nagasaki, T., Iwamoto, K., Ikeda, A., He, G. X., Matsuda, T., and Iwamoto, M. (1991) New syntheses and physical properties of para-alkylcalix[n]arenes. Bull. Chem. Soc. Jpn. 64, 381− 386. (31) Chen, F. C. M., and Benoiton, N. L. (1976) A new method of quaternizing amines and its use in amino acid and peptide chemistry. Can. J. Chem. 54, 3310−3311. (32) Shinkai, S., Arimura, T., Araki, K., Kawabata, H., Satoh, H., Tsubaki, T., Manabe, O., and Sunamoto, J. (1989) Syntheses and aggregation properties of new water-soluble calixarenes. J. Chem. Soc., Perkin Trans. 1, 2039−2045. (33) Helttunen, K., and Shahgaldian, P. (2010) Self-assembly of amphiphilic calixarenes and resorcinarenes in water. New J. Chem. 34, 2704−2714. (34) Rodik, R. V., Klymchenko, A. S., Jain, N., Miroshnichenko, S. I., Richert, L., Kalchenko, V. I., and Mely, Y. (2011) Virus-sized DNA nanoparticles for gene delivery based on micelles of cationic calixarenes. Chem.Eur. J. 17, 5526−5538. (35) Zinchenko, A. A., Pyshkina, O. A., Lezov, A. V., Sergeyev, V. G., and Yoshikawa, K. (2008) Single DNA Molecules: Compaction and Decompaction, DNA Interactions with Polymers and Surfactants (Dias, R. S., and Lindman, B., Eds.) pp 59−88, John Wiley & Sons, Inc., Hoboken, NJ. (36) Golan, R., Pietrasanta, L. I., Hsieh, W., and Hansma, H. G. (1999) DNA toroids: stages in condensation. Biochem. 38, 14069− 14076.

supported this research, and the Centro Interdipartimentale Misure “G. Casnati” for the use of NMR, HRMS, and AFM facilities. This work was carried out in the frame of COST Actions CM1005 and CM1102.

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REFERENCES

(1) Mintzer, M. A., and Simanek, E. E. (2009) Nonviral vectors for gene delivery. Chem. Rev. 109, 259−302. (2) Bhattacharya, S., and Bajaj, A. (2009) Advances in gene delivery through molecular design of cationic lipids. Chem. Commun., 4632− 4656. (3) Srinivas, R., Samanta, S., and Chaudhuri, A. (2009) Cationic amphiphiles: promising carriers of genetic materials in gene therapy. Chem. Soc. Rev. 38, 3326−3338. (4) Zuhorn, I. S., Engberts, J. B. F. N., and Hoekstra, D. (2007) Gene delivery by cationic lipid vectors: overcoming cellular barriers. Eur. Biophys. J. 36, 349−362. (5) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection - A highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413−7417. (6) Dass, C. R. (2004) Lipoplex-mediated delivery of nucleic acids: factors affecting in vivo transfection. J. Mol. Med. Immunol. 82, 579− 591. (7) Boussif, O., Lezoualch, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297−7301. (8) Merdan, T., Kopecek, J., and Kissel, T. (2002) Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv. Drug Delivery Rev. 54, 715−758. (9) Sizovs, A., McLendon, P. M., Srinivasachari, S., and Reineke, T. M. (2010) Carbohydrate polymers for nonviral nucleic acid delivery. Top. Curr. Chem. 296, 131−190. (10) Haensler, J., and Szoka, F. C. J. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 4, 372−379. (11) Bielinska, A. U., Chen, C., Johnson, J., and Baker, J. R. J. (1999) DNA complexing with polyamidoamine dendrimers: Implications for transfection. Bioconjugate Chem. 10, 843−850. (12) Guillot-Nieckowski, M., Eisler, S., and Diederich, F. (2007) Dendritic vectors for gene transfection. New J. Chem. 31, 1111−1127. (13) Bertling, W. M., Gareis, M., Paspaleeva, V., Zimmer, A., Kreuter, J., Nuernberg, E., and Harrer, P. (1991) Use of liposomes, viral capsids, and nanoparticles as DNA carriers. Biotechnol. Appl. Biochem. 13, 390−405. (14) Sandhu, K. K., McIntosh, C. M., Simard, J. M., Smith, S. W., and Rotello, V. M. (2002) Gold nanoparticle-mediated transfection of mammalian cells. Bioconjugate Chem. 13, 3−6. (15) Aoyama, Y. (2004) Macrocyclic glycoclusters: from amphiphiles through nanoparticles to glycoviruses. Chem.Eur. J. 10, 588−593. (16) Sansone, F., Dudic, M., Donofrio, G., Rivetti, C., Baldini, L., Casnati, A., Cellai, S., and Ungaro, R. (2006) DNA condensation and cell transfection properties of guanidinium calixarenes: Dependence on macrocycle lipophilicity, size, and conformation. J. Am. Chem. Soc. 128, 14528−14536. (17) Ortiz Mellet, C., Benito, J. M., and García Fernández, J. M. (2010) Preorganized, macromolecular, gene-delivery systems. Chem. Eur. J. 16, 6728−6742. (18) Ortiz Mellet, C., García Fernández, J. M., and Benito, J. M. (2011) Cyclodextrin-based gene delivery systems. Chem. Soc. Rev. 40, 1586−1608. (19) Srinivasachari, S., Fichter, K. M., and Reineke, T. M. (2008) Polycationic beta-cyclodextrin “click clusters”: monodisperse and versatile scaffolds for nucleic acid delivery. J. Am. Chem. Soc. 130, 4618−4627. (20) Mourtzis, N., Paravatou, M., Mavridis, I. M., Roberts, M. L., and Yannakopoulou, K. (2008) Synthesis, characterization, and remarkable 1001

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Bioconjugate Chemistry

Article

(37) Sternberg, B., Sorgi, F. L., and Huang, L. (1994) New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy. FEBS Lett. 356, 361−366. (38) Pitard, B., Aguerre, O., Airiau, m., Lachages, A. M., Boukhnikachvili, T., Byk, G., Dubertret, C., Herviou, C., Scherman, D., Mayaux, J. F., and Crouzet, J. (1997) Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer. Proc. Natl. Acad. Sci. U.S.A. 94, 14412−14417. (39) Nakai, T., Kanamori, T., Sando, S., and Aoyama, Y. (2003) Remarkably size-regulated cell invasion by artificial viruses. saccharidedependent self-aggregation of glycoviruses and its consequences in glycoviral gene delivery. J. Am. Chem. Soc. 125, 8465−8475. (40) The discrepancy between the percentages of transfected cells of the Vero line reported here with respect to those indicated in ref 25 is due to the different clone used in the new experiments performed for this article. However, the substantial accordance found now and before in the gap (10%) between the efficiency of LTX and 5b/DOPE confirms the data reproducibility and goodness. (41) Diaz-Moscoso, A., Vercauteren, D., Rejman, J., Benito, J. M., Mellet, C. O., De Smedt, S. C., and Fernandez, J. M. G. (2010) Insights in cellular uptake mechanisms of pDNA-polycationic amphiphilic cyclodextrin nanoparticles (CDplexes). J. Controlled Release 143, 318−325. (42) No significant deprotonation of guanidium groups directly linked at the upper rim of a calix[4]arene was observed till pH 8 in titration experiments. Baldini, L., Cacciapaglia, R., Casnati, A., Mandolini, L., Salvio, R., Sansone, F., and Ungaro, R. (2012) Upper Rim Guanidinocalix[4]arenes as Artificial Phosphodiesterases. J. Org. Chem. 77, 3381−3389. (43) Hansma, H. G., Golan, R., Hsieh, W., Lollo, C. P., Mullen-Ley, P., and Kwoh, D. (1998) DNA condensation for gene therapy as monitored by atomic force microscopy. Nucleic Acids Res. 26, 2481− 2487. (44) Lalor, R., Di Gesso, J. L., Mueller, A., and Matthews, S. E. (2007) Efficient gene transfection with functionalised multicalixarenes. Chem. Commun., 4907−4909.

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dx.doi.org/10.1021/bc2006829 | Bioconjugate Chem. 2012, 23, 993−1002