8478
Langmuir 2006, 22, 8478-8484
Supramolecular Assembly of Cyclodextrin-Based Nanoparticles on Solid Surfaces for Gene Delivery In-Kyu Park,† Horst A. von Recum,†,‡ Shaoyi Jiang,| and Suzie H. Pun*,† Departments of Bioengineering and Chemical Engineering, UniVersity of Washington, Seattle, Washington 98195 ReceiVed June 19, 2006. In Final Form: July 12, 2006 In this work, a new approach for surface-mediated gene delivery based on inclusion complex formation between the solid surface and delivery vehicles is presented. β-Cyclodextrin (CD) molecules form high-affinity inclusion complexes with adamantane. This complexation ability was used to specifically immobilize β-CD-modified poly(ethylenimine) (CD-PEI) nanoparticles on adamantane- (AD-) modified self-assembled monolayers. To investigate the nanoparticle/surface interaction, CD-PEI-based and PEI-based nanoparticles were passed through a surface plasmon resonance flow cell containing the monolayers. CD-PEI nanoparticles are specifically immobilized on the chip surface by cyclodextrin-adamantane inclusion complex formation. Minimal nanoparticle adsorption was detected with PEIbased nanoparticles or on control surfaces. Competition studies with free cyclodextrins reveal that the multivalent interactions between CD-PEI nanoparticles and the adamantane-modified surface results in significantly higher binding affinity than single cyclodextrin-adamantane complexes. Immobilized nanoparticles were characterized by atomic force microscopy and quantified by fluorescence assay. Thus, the ability of CD-PEI nanoparticles to form inclusion complexes can be exploited to attain specific, high-affinity loading of delivery vehicles onto solid surfaces.
Introduction One approach for localized delivery of nucleic acids is immobilization of delivery vehicles onto solid surfaces that are then placed in contact with cells.1,2 This technology has important applications ranging from gene therapy to tissue engineering to functional genomics.3 For example, gene therapy can be combined with implanted biomedical devices to improve integration with host tissue,4,5 and nucleic acid delivery vehicles can be integrated with engineered matrices to promote functional tissue growth.6 Nonviral gene delivery vehicles have been immobilized to solid surfaces by physical adsorption,7,8 biotin-streptavidin interaction,9,10 and chemical conjugation.11,12 Here, we present a new approach for surface-mediated gene delivery based on supramolecular assembly of delivery vehicles on functionalized surfaces. * To whom correspondence should be addressed: e-mail spun@ u.washington.edu; tel (206) 685-3488; fax (206) 616-3928. † Department of Bioengineering. ‡ Present address: Biomedical Engineering, Case Western University, Cleveland, OH. | Department of Chemical Engineering. (1) Shea, L. D.; Smiley, E.; Bonadio, J.; Mooney, D. J. DNA delivery from polymer matrices for tissue engineering. Nat. Biotechnol. 1999, 17 (6), 551-554. (2) Luo, D.; Saltzman, W. M. Enhancement of transfection by physical concentration of DNA at the cell surface. Nat. Biotechnol. 2000, 18 (8), 893-895. (3) Pannier, A. K.; Shea, L. D. Controlled release systems for DNA delivery. Mol. Ther. 2004, 10 (1), 19-26. (4) Klugherz, B. D.; Song, C. X.; Defelice, S.; Cui, X. M.; Lu, Z. B.; Connolly, J.; Hinson, J. T.; Wilensky, R. L.; Levy, R. J. Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Hum. Gene Ther. 2002, 13 (3), 443-454. (5) Sharif, F.; Daly, K.; Crowley, J.; O’Brien, T. Current status of catheterand stent-based gene therapy. CardioVasc. Res. 2004, 64 (2), 208-216. (6) Jang, J. H.; Houchin, T. L.; Shea, L. D. Gene delivery from polymer scaffolds for tissue engineering. Expert ReV. Med. DeVices 2004, 1 (1), 127-138. (7) Bielinska, A. U.; Yen, A.; Wu, H. L.; Zahos, K. M.; Sun, R.; Weiner, N. D.; Baker, J. R.; Roessler, B. J. Application of membrane-based dendrimer/DNA complexes for solid-phase transfection in vitro and in vivo. Biomaterials 2000, 21 (9), 877-887. (8) Bengali, Z.; Pannier, A. K.; Segura, T.; Anderson, B. C.; Jang, J. H.; Mustoe, T. A.; Shea, L. D. Gene delivery through cell culture substrate adsorbed DNA complexes. Biotechnol. Bioeng. 2005, 90 (3), 290-302. (9) Segura, T.; Volk, M. J.; Shea, L. D. Substrate-mediated DNA delivery: role of the cationic polymer structure and extent of modification. J. Controlled Release 2003, 93 (1), 69-84. (10) Segura, T.; Shea, L. D. Surface-tethered DNA complexes for enhanced gene delivery. Bioconjugate Chem. 2002, 13 (3), 621-629.
Cyclodextrins (CDs) are cyclic oligomers of six, seven, or eight glucose molecules; β-cyclodextrin contains seven glucose molecules in a toroidal structure (Scheme 1A).13 The outside of the β-cyclodextrin toroid is hydrophilic, imparting the molecules with high water solubility, and the interior of the toroid is relatively hydrophobic. As a result, CDs form inclusion complexes, acting as “hosts” to hydrophobic “guest” molecules that reside within the toroid. For example, adamantane forms inclusion complexes with β-cyclodextrins with high association constants on the order of 104-105 (Scheme 1B).14 This high affinity has been exploited in applications such as biosensing,15-17 drug delivery,18,19 selfassembled patterning for nanofabrication,20-22 and chromatography.23 Cyclodextrin-based polycations have been synthesized for nucleic acid delivery.24,25 When these are mixed with nucleic acids, the polymers and nucleic acids self-assemble by electrostatic (11) Trentin, D.; Hubbell, J.; Hall, H., Nonviral gene delivery for local and controlled DNA release. J. Controlled Release 2005, 102 (1), 263275. (12) Zheng, J.; Manuel, W. S.; Hornsby, P. J. Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine. Biotechnol. Prog. 2000, 16 (2), 254-257. (13) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1988; p 464. (14) Cromwell, W.; Bystrom, K.; Eftink, M. Cyclodextrin adamantanecarboxylate inclusion complexes: studies of the variation in cavity size. J. Phys. Chem. 1985, 89, 326-332. (15) Wintgens, V.; Amiel, C. Surface plasmon resonance study of the interaction of a β-cyclodextrin polymer and hydrophobically modified poly(N-isopropylacrylamide). Langmuir 2005, 21 (24), 11455-11461. (16) Fragoso, A.; Caballero, J.; Almirall, E.; Villalonga, R.; Cao, R. Immobilization of adamantane-modified cytochrome c at electrode surfaces through supramolecular interactions. Langmuir 2002, 18 (13), 5051-5054. (17) David, C.; Herve, F.; Sebille, B.; Canva, M.; Millot, M. C. The reversible binding of immunoglobulins G modified with adamantyl-end-capped poly(ethylene glycol)s to poly-beta-cyclodextrin-coated gold surfaces and their interactions with specific target molecules: A surface plasmon resonance investigation. Sens. Actuators, B 2006, 114 (2), 869-880. (18) Pun, S.; Bakker, A.; Bellocq, N.; Grubbs, B.; Jensen, G.; Liu, A.; Cheng, J.; Janssens, B.; Floren, W.; Peeters, J.; Janicot, M.; Davis, M.; Brewster, M. Biodistribution of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles. Cancer Biol. Ther. 2004, 3 (7), 641-650. (19) Bellocq, N. C.; Kang, D. W.; Wang, X. H.; Jensen, G. S.; Pun, S. H.; Schluep, T.; Zepeda, M. L.; Davis, M. E. Synthetic biocompatible cyclodextrinbased constructs for local gene delivery to improve cutaneous wound healing. Bioconjugate Chem. 2004, 15 (6), 1201-1211.
10.1021/la061757s CCC: $33.50 © 2006 American Chemical Society Published on Web 08/24/2006
Cyclodextrin-Based Nanoparticles for Gene DeliVery
Langmuir, Vol. 22, No. 20, 2006 8479
Scheme 1. (A) Structure of β-Cyclodextrin, (Β) Schematic of Adamantane and β-Cyclodextrin Inclusion Complex Formation, and (C) Schematic of Cyclodextrin Nanoparticle Immobilization on Adamantane-Modified Surfaces by Inclusion Complex Formation
interactions and condense into nanoparticulate structures called “polyplexes”. These nanoparticles are capable of transfecting cultured cells with high efficiency and relatively low toxicity.26-29 In addition, cyclodextrins on the surface of these nanoparticles are capable of forming inclusion complexes with hydrophobic guest molecules.30,31 Formulation of cyclodextrin-based nanoparticles with adamantane-poly(ethylene glycol) conjugates results in salt stabilization of the nanoparticles due to poly(ethylene glycol) attachment to the nanoparticle surface by adamantane-cyclodextrin interaction.31 In this work, nanoparticle complexes of cyclodextrin polycations and nucleic acids were specifically immobilized on adamantane-functionalized surfaces by inclusion complex formation (Scheme 1C). One major advantage of this method is that (20) Mahalingam, V.; Onclin, S.; Peter, M.; Ravoo, B. J.; Huskens, J.; Reinhoudt, D. N. Directed self-assembly of functionalized silica nanoparticles on molecular printboards through multivalent supramolecular interactions. Langmuir 2004, 20 (26), 11756-11762. (21) Banerjee, I. A.; Yu, L. T.; Matsui, H. Application of host-guest chemistry in nanotube-based device fabrication: Photochemically controlled immobilization of azobenzene nanotubes on patterned R-CD monolayer/Au substrates via molecular recognition. J. Am. Chem. Soc. 2003, 125 (32), 9542-9543. (22) Huskens, J.; Deij, M. A.; Reinhoudt, D. N. Attachment of molecules at a molecular printboard by multiple host-guest interactions. Angew. Chem., Int. Ed. 2002, 41 (23), 4467. (23) Karakasyan, C.; Millot, M. C.; Vidal-Madjar, C. Immobilization of a (dextran-adamantane-COOH) polymer onto beta-cyclodextrin-modified silica. J. Chromatogr., B 2004, 808 (1), 63-67. (24) Davis, M. E.; Pun, S. H.; Bellocq, N. C.; Reineke, T. M.; Popielarski, S. R.; Mishra, S.; Heidel, J. D. Self-assembling nucleic acid delivery vehicles via linear, water-soluble, cyclodextrin-containing polymers. Curr. Med. Chem. 2004, 11 (2), 179-197. (25) Pun, S. H.; Davis, M. E. Cyclodextrin polymers for gene delivery. In Polymeric Gene DeliVery; Amiji, M., Ed.; CRC Press: Boca Raton, FL, 2005; pp 187-210. (26) Kihara, F.; Arima, H.; Tsutsumi, T.; Hirayama, F.; Uekama, K. In vitro and in vivo gene transfer by an optimized R-cyclodextrin conjugate with polyamidoamine dendrimer. Bioconjugate Chem. 2003, 14 (2), 342-350. (27) Hwang, S.; Bellocq, N.; Davis, M. Effects of Structure of β-CyclodextrinContaining Polymers on Gene Delivery. Bioconjugate Chem. 2001, 12 (2), 280290. (28) Gonzalez, H.; Hwang, S.; Davis, M. New class of polymers for the delivery of macromolecular therapeutics. Bioconjugate Chem. 1999, 10, 1068-74. (29) Popielarski, S. R.; Mishra, S.; Davis, M. E. Structural effects of carbohydrate-containing polycations on gene delivery. 3. CD type and functionalization. Bioconjugate Chem. 2003, 14, 672-678. (30) Forrest, M. L.; Gabrielson, N.; Pack, D. W. Cyclodextrin-polyethylenimine conjugates for targeted in vitro gene delivery. Biotechnol. Bioeng. 2004, 89, 416-423. (31) Pun, S. H.; Davis, M. Development of a Non-Viral Gene Delivery Vehicle for Systemic Application. Bioconjugate Chem. 2002, 13 (3), 630-639.
guest molecules grafted on the surfaces results in high nanoparticle binding with minimal changes in surface properties such as hydrophilicity and charge. In addition, because the immobilization occurs by inclusion complex formation, the functionalized surfaces can be readily synthesized and characterized. Nanoparticle immobilization then takes place by self-assembly on the surfaces without additional need for chemical conjugation and added reagents. The nanoparticles are held on the surface by multivalent inclusion complex formation and are therefore stably immobilized but can also be reversibly released for cellular uptake. Here, adamantane-modified surfaces were prepared by conjugation of activated adamantanes with self-assembled monolayers on gold displaying terminal amines. CD-nanoparticle immobilization on these surfaces is monitored by surface plasmon resonance (SPR) spectroscopy and characterized by atomic force microscopy (AFM). Experimental Section Materials. pGL3-CV plasmid was purchased from Promega Corp. (Madison, WI) and amplified under endotoxin-free conditions by Elim Biopharmaceuticals (Hayward, CA). PicoGreen reagent was purchased from Invitrogen (Carlsbad, CA). 6-Monotosyl-β-cyclodextrin was purchased from CTD, Inc. (Gainesville, FL). (1Mercaptoundec-11-yl)tetra(ethylene glycol), HS-(CH2)11-EG4, and (1-mercaptoundec-11-yl)hexa(ethylene glycol)amine, HS-(CH2)11EG6-NH2, were purchased from Prochimia (Sopot, Poland). 1-Adamantanecarboxylic acid was purchased from Acros Organics. Poly(2-ethyl-2-oxazoline) and other chemicals and solvents were purchased from Sigma-Aldrich. AFM mica disks were purchased from Ted Pella, Inc. (Redding, CA). Synthesis of Cyclodextrin-Modified, Linear Poly(ethylenimine)). Linear PEI was synthesized by acid hydrolysis of poly(2-ethyl-2-oxazoline) via a protocol provided by Ernst Wagner (LMU Munich, Germany). In brief, poly(2-ethyl-2-oxazoline) (200 kDa) was deacylated with 24% hydrochloric acid at 105 °C under reflux for 6 days. The resulting deacylated linear PEI was neutralized with 5 N NaOH and then dialyzed extensively against water in a Spectra/ Por molecular weight cutoff (MWCO) 10K membrane. The product was lyophilized and complete removal of oxazoline group was verified by NMR. CD-PEI was synthesized from the deacylated PEI according to a modified, published procedure.32 Deacetylated, linear (32) Pun, S.; Bellocq, N.; Liu, A.; Davis, M. Cyclodextrin-modified polyethylenimine polymers for gene delivery. Bioconjugate Chem. 2004, 15, 831840.
8480 Langmuir, Vol. 22, No. 20, 2006 PEI (200 mg, 8 µmol) was dissolved in 14.4 mL of dimethyl sulfoxide (DMSO) and heated to 70 °C. 6-Monotosyl-β-cyclodextrin (1.561 g, 1.2 mmol) was added in small aliquots and the resulting mixture was stirred at 70 °C for 6 days. Hydroxylamine (1 mL, 18 mmol) was then added to inactivate unreacted cyclodextrins. Water (45 mL) was added to the solution which was dialyzed against water in a Spectra/Por MWCO 3500 membrane for 3 days. The precipitate, consisting of unreacted cyclodextrins and linear PEI with low grafting ratios, was removed by filtration. The desired polymer was further purified by ultrafiltration through a stirred cell with MWCO 10K. The filtered solution was lyophilized to obtain a fluffy white solid in 17.6% yield. The grafting ratio of the polymer was determined by proton integration of 1H NMR. The integrated peak area at 5.08 ppm, corresponding to C1H of cyclodextrin, was compared to the integrated peak area at 2.5-3.2 ppm, corresponding to ethylenimine. A grafting ratio of 1 cyclodextrin per 9 ethylenimine repeat units was achieved. Nanoparticle Formulation and Characterization. CD-PEI and PEI nanoparticles (“polyplexes”) were prepared by adding an equal volume of polymer to pGL3-CV plasmid (0.125 mg/mL in H2O) at a 5 polymer nitrogen to 1 DNA phosphate ratio. Polymers were prepared as a stock concentration at 1 mg/mL at pH 7.0 and diluted in distilled water to the desired concentration before being mixed with DNA. Nanoparticle size and surface charge was measured by a ZetaPALS dynamic light scattering detector (Brookhaven Instruments Corp., Holtsville, NY) in five replicates. Dynamic light scattering measurements were conducted with a 90° detection angle at 659 nm. Formation of Self-Assembled Monolayers and AdamantaneModified SAMs. Gold substrates were prepared by coating BK-7 glass substrates with a 2 nm layer of chromium followed by a 50 nm layer of gold by electron-beam evaporation. The substrates were then cleaned by rinsing with ethanol and water, drying under a nitrogen stream, treating under UV/ozone for 20 min, and rinsing again with deionized water and ethanol. The chips were immediately immersed in 90% ethanol solutions containing 100 µM each of HS-(CH2)11-EG4 and HS-(CH2)11-EG6-NH2 and 3% (v/v) ammonium hydroxide for 24 h at -20 °C for SAM formation, followed by rinsing with ethanol, 10% (v/v) acetic acid in ethanol, and ethanol. 1-Adamantanecarboxylic acid or 3-(1,1,2,3,3,3-hexafluoropropyl)adamantane-1-carboxylic acid was converted to the activated ester by dissolving the adamantane compound in dimethyl formamide (DMF) at 10 mM concentration and then adding N-hydroxysuccinimide (50 mM in DMF) and dicyclohexylcarbodiimide (50 mM in DMF) at 5 molar equiv to adamantane. The solution was stirred for 24 h at 4 °C and then centrifuged to remove the insoluble dicyclohexylurea byproduct. The activated adamantane ester was then added to the SAMs at 1 mM concentration for 24 h at 4 °C. The AD-SAM was washed three times in ethanol, followed by drying under a stream of nitrogen. Before use, AD-SAMs were washed again by rinsing with ethanol, 10% (v/v) acetic acid in ethanol, and ethanol. XPS measurements were taken on a Surface Science Instruments X-probe spectrometer, which is equipped with a monochromatized Al KR X-ray and a low-energy electron flood gun for charge neutralization. The pass energy for survey spectra was 150 eV and takeoff angle was 55°. The Service Physics ESCAVB Graphics Viewer program was used to determine peak areas. Three X-ray spots (∼800 µm) were taken for each sample and reported as the mean value. The actual surface compositions of mixed HS-(CH2)11EG4 and HS-(CH2)11-EG6-NH2 SAM were calculated on the basis of N and S signals by comparing with the composition of monolayers formed from the pure thiols. For AD-SAM, the multifluorinated adamantane was used to estimate AD composition, which is calculated by comparing the F signal with N and O signals. Contact angle of AD-SAM was measured by the static drop method. A drop of water was placed on the surface of AD-SAM, and the contact angle at the interface between water and the AD-SAM surface was measured with a goniometer under ambient laboratory conditions. SPR Instrumentation and Measurements. A custom-built dualchannel SPR sensor based on the Kretschmann configuration of the
Park et al. attenuated total reflection (ATR) method was used in this work. It utilizes wavelength interrogation and incorporates two parallel flow channels. The details of the instrument setup and operation principles were described previously.33,34 A functionalized SPR chip is attached to the base of an optical prism from the glass side mediated with a refractive index matching fluid (Cargille Labs, Cedar Groves, NJ) while the metal side is mechanically pressed against an acrylic manifold with two parallel flow cells. A polychromatic light beam passes through the optical prism and the glass substrate at a fixed angle and excites a surface plasmon wave at the dielectric-metal interface. The reflected light from the areas corresponding to two flow cells is detected separately by two optical spectrographs (Ocean Optics, Dunedin, FL). The excitation of the surface plasmon is accompanied by the transfer of optical energy into surface plasmon and dissipation in the metal layer, resulting in a narrow dip in the spectrum of reflected light. The wavelength at which the resonant excitation of the surface plasmon occurs depends on the refractive index of the analyte in the proximity to the SPR surface. As the refractive index increases, the resonant wavelength shifts to high values. The wavelength shift was used to measure the change in surface concentration (mass per unit area). Thus, an SPR sensorgram is a plot of resonant wavelength shift versus time, giving the amount of analyte binding as a function of time. The SPR chip is a glass substrate coated with a 2 nm adhesion layer of chromium followed by a 50 nm surface-plasmon-active layer of gold by electron beam evaporation. Surface functionalization was described above. A liquid sample is delivered to the sensor surface through an acrylic flow cell manifold by use of a peristaltic pump (Ismatec, Glattbrugg, Switzerland). The acrylic flow cell manifold contains two independent parallel inlets and outlets. A Mylar gasket 50 µm thick is attached to the manifold, creating two independent parallel flow channels. All buffers and solutions were degassed with vacuum for 30 min before analyses. For nanoparticle immobilization studies, the SPR was stabilized with water at room temperature for 5 min. Nanoparticle solutions were then injected into the flow cell for 15 min by use of a peristaltic pump, followed by washing with water to remove loosely associated material. For maltoheptaose and β-cyclodextrin competition experiments, the SPR chip was washed with water for 5 min. The maltoheptaose or β-cyclodextrin solution (10 mM in distilled H2O) was then flowed through for 10 min, followed by another 10 min wash with water. The CD-PEI nanoparticles solution was then added for 20 min followed by addition of β-cyclodextrin solution (10 mM) for another 10 min. All solutions were introduced at 50 µL/min flow rate. Each run was then ended with a final 10 min wash with water. Quantitation of Nanoparticles Bound on Monolayers. SAM and AD-SAM were formed on gold-coated glass substrates as described above. CD-PEI and PEI nanoparticles were formulated as described above and incubated with substrates for 2 h at 4 °C. Substrates were then washed several times with distilled water and dried under a stream of air. Plasmid DNA was released from monolayer surfaces by incubation of 0.3 mL of heparan sulfate solution (0.167 mg/mL) with substrates for 30 min at 4 °C. Solutions were then collected and the amount of released DNA was quantified by the PicoGreen Assay according to the manufacturer’s instructions. Released DNA was quantified by converting fluorescence (excitation 480 nm, emission 520 nm) to DNA content by use of a standard curve prepared with known concentrations of CD-PEI and PEI nanoparticles treated with heparan sulfate (standard curves showed a linear relationship between fluorescence and DNA concentration with R ) 0.9992). Atomic Force Microscopic Imaging of Nanoparticles. AFM substrates were prepared by coating AFM mica disks with gold (3 nm Cr/50 nm Au). SAM and AD-SAM were prepared on the goldcoated mica disks as described in preceding sections. PEI and CDPEI nanoparticles were added to the substrates by pipetting 0.1 mL (33) Homola, J.; Dostalek, J.; Chen, S. F.; Rasooly, A.; Jiang, S. Y.; Yee, S. S. Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk. Int. J. Food Microbiol. 2002, 75 (1-2), 61-69. (34) Homola, J., Present and future of surface plasmon resonance biosensors. Anal. Bioanal. Chem. 2003, 377 (3), 528-539.
Cyclodextrin-Based Nanoparticles for Gene DeliVery
Langmuir, Vol. 22, No. 20, 2006 8481
Figure 1. (Left) PEI and CD-PEI nanoparticles are formed by condensation of plasmid DNA with PEI and CD-PEI, respectively, at a polymer amine:DNA phosphate ratio of 5. (Right) The PEI and CD-PEI nanoparticles have similar particle size and surface charge, as determined by dynamic light scattering measurements.
Figure 2. Preparation of amine-terminated, self-assembled monolayers (SAMs) and adamantane-functionalized, self-assembled monolayers (AD-SAMs). Adamantane was conjugated to SAMs by DCC coupling reaction. of nanoparticles on the surface of each mica disk and incubating for 10 s at room temperature. The disks were then washed twice with distilled water and dried with filter paper. The AFM images were obtained in tapping mode by using Digital Instruments Nanoscope III equipped with a 15 µm scanner. A commercial silicon cantilever (Otespa, Veeco) with resonant frequencies ranging from 231 to 320 kHz was used. Images were processed with Nanoscope III software and expressed in amplitude mode.
Results and Discussion Cyclodextrin-modified, linear PEI (CD-PEI) was used as the nonviral gene carrier in these studies because our previous work demonstrated high gene transfer efficiencies with this polymer.32 Deacylated PEI was prepared by acid hydrolysis of poly(2-ethyl2-oxazoline); fully deacylated PEI has been shown to provide superior nucleic acid delivery over commercially available linear PEI.35 CD-PEI was synthesized by reaction of activated,
monotosyl-β-cyclodextrin with deacylated linear PEI according to published procedures.32 The grafting ratio was determined by 1H NMR to be 1 CD per 9 ethylenimine repeat units. Deactylated, linear PEI was used in control formulations. CD-PEI nanoparticles and control PEI nanoparticles were formulated by mixing polymer with plasmid DNA at nitrogen:phosphate ratios of 5 in distilled water. The polymer and DNA self-assemble and condense to uniform nanoparticles (Figure 1, left). The particle size and surface charge were measured by dynamic light scattering. Both CDPEI and PEI nanoparticles have similar size (∼100 nm), polydispersity (0.129 ( 0.047 for CD-PEI nanoparticles and 0.139 ( 0.023 for PEI nanoparticles), and surface charge (∼+10 mV) (Figure 1, right). Previous studies have demonstrated by (35) Thomas, M.; Lu, J. J.; Ge, Q.; Zhang, C. C.; Chen, J. Z.; Klibanov, A. M. Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (16), 5679-5684.
8482 Langmuir, Vol. 22, No. 20, 2006
Park et al.
Table 1. Composition and Contact Angle Characterization of SAM and AD-SAM SAM SAM AD-SAM
content by XPS, %
contact angle (SD), deg
HS-C11-EG4-OH, 73.5; HS-C11-EG4-NH2, 26.5; HS-C11-EG4-AD, 0 HS-C11-EG4-OH, 73.5; HS-C11-EG4-NH2, 22.0; HS-C11-EG4-AD, 4.5
22.9 (1.5) 23.8 (1.1)
electron microscopy that the nanoparticles are uniform and spherical in shape when formulated in water.32,36 Amine-terminated, self-assembled monolayers (SAMs) were prepared by adding solutions of HS-(CH2)11-EG4 and HS-(CH2)11EG6-NH2 in 90% ethanol to gold-coated glass coverslips in the presence of a base for SAM formation, followed by rinsing with 10% acetic acid in ethanol. This procedure has been shown to produce high-quality, single-layer SAMs by disrupting hydrogen bonds between the surface-bound and solution-phase thiols.37,38 The long-chain HS(CH2)11(C2H5O)6NH2 thiols, protruding from the nonfouling background of the short-chain HS(CH2)11(C2H5O)4OH thiols, were used to tether adamantane and to avoid steric effects when CD-PEI nanoparticles bind to the adamantane immobilized surface.39 Hexa(ethylene glycol)-terminated SAMs resist protein adsorption and amine-functionalized surfaces can be protonated to minimize nanoparticle adsorption by charge repulsion, since CD-PEI and PEI nanoparticles are formulated with an overall positive surface charge. The amine-terminated SAMs were converted to adamantane-modified SAMs (ADSAMs) by reaction with activated esters of adamantane (Figure 2). 1-Adamantanecarboxylic acid was reacted with dicyclo-
Figure 4. Competition studies with β-cyclodextrin (top) and maltoheptaose (bottom) on AD-SAM. Free sugars were added at 5 min and surfaces were washed with water at 15 min for 10 min. CD-PEI nanoparticles were introduced at 25 min for 20 min, and then surfaces washed again with water for 10 min. Solutions of 10 mM β-cyclodextrin were again added at 60 min for 20 min followed by washing with water. β-Cyclodextrin interacts with AD-SAM, whereas the linear maltoheptaose does not. CD-PEI nanoparticles are not displaced by 10 mM β-cyclodextrin (greater than 10 000fold excess to adamantanes), demonstrating high binding constants to the AD-SAM due to multivalent interactions.
hexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), and insoluble byproducts were removed by precipitation. The activated adamantane molecules were reacted with SAM for 24 h, followed by extensive washing to remove unreacted materials. To characterize the SAMs by X-ray photoelectron spectroscopy (XPS), AD-SAMs were also prepared from hexafluoroadamantanes; the fluorine atoms provide a sensitive tag for quantifying adamantane content. The surface composition of SAM and ADSAM was determined by XPS measurements (Table 1). The fraction of amine-terminated versus hydroxyl-terminated thiols in the SAM was calculated by comparing the N and S signals from the mixed SAM to those from pure SAMs. Both monolayers were composed of ∼25% amine and ∼75% hydroxyl termini.
Figure 3. (Top) SPR sensorgrams for PEI (---) and CD-PEI (s) nanoparticle interaction with unmodified SAM. (Bottom) SPR sensorgrams for PEI (---) and CD-PEI (s) nanoparticle interaction with AD-SAM. Nanoparticles were added at 5 min and surfaces were washed with water at 20 min.
(36) Tang, M.; Szoka, J. The Influence of Polymer Structure on the Interactions of Cationic Polymers with DNA and Morphology of the Resulting Complexes. Gene Ther. 1997, 4, 823-832. (37) Wang, H.; Chen, S. F.; Li, L. Y.; Jiang, S. Y. Improved method for the preparation of carboxylic acid and amine terminated self-assembled monolayers of alkanethiolates. Langmuir 2005, 21 (7), 2633-2636. (38) Li, L. Y.; Chen, S. F.; Zheng, J.; Ratner, B. D.; Jiang, S. Y. Protein adsorption on oligo(ethylene glycol)-terminated alkanethiolate self-assembled monolayers: The molecular basis for nonfouling behavior. J. Phys. Chem. B 2005, 109 (7), 2934-2941. (39) Yu, Q. M.; Chen, S. F.; Taylor, A. D.; Homola, J.; Hock, B.; Jiang, S. Y. Detection of low-molecular-weight domoic acid using surface plasmon resonance sensor. Sens. Actuators, B 2005, 107 (1), 193-201.
Cyclodextrin-Based Nanoparticles for Gene DeliVery
Langmuir, Vol. 22, No. 20, 2006 8483
Figure 5. (Top) Schematic of DNA quantification by heparan sulfate displacement of plasmid DNA from immobilized nanoparticles. (Bottom) Comparison of the amount of DNA immobilized by CD-PEI on AD-SAM, by PEI on AD-SAM, and by CD-PEI on SAM. Data were collected in three separate experiments and reported as mean with standard deviation. *p < 0.01 compared with control samples.
Adamantane was conjugated to ∼17% of amine termini in ADSAM (4.5% of total composition was HS-C11-EG6-AD). Multifluorinated adamantane was used to determine adamantane composition. The F signal was compared to N and O signals in AD-SAM. The relative hydrophobicity of the monolayers was determined by contact angle measurements. Functionalization of SAMs with adamantane has no significant effect on surface hydrophobicity (contact angle of 22.9° ( 1.5° on SAM and 23.8° ( 1.1° on AD-SAM). The interaction of nanoparticles with the monolayers was investigated by SPR spectroscopy. In the SPR configuration used in this study, nanoparticle adsorption to the sensor surface is detected by a shift in resonance wavelength. An SPR sensorgram is a plot of resonant wavelength shift versus time, giving the amount of analyte binding as a function of time. SAM surfaces lacking immobilized guest molecules served as control monolayers, while PEI nanoparticles that lacked displayed host molecules were used as control particles. PEI and CD-PEI nanoparticles interacted minimally with SAM surfaces, although some slow nonspecific adsorption was observed with time (Figure 3, top). PEI nanoparticles similarly showed no interaction with AD-SAM surfaces due to the nonfouling OEG background and to electrostatic repulsion between the cationic particles and unreacted amines (Figure 3, bottom). In contrast, CD-PEI nanoparticles are specifically immobilized on the AD-SAM surface. No significant nanoparticle desorption is observed after washing the sensor surface with water, suggesting significant association constants between CD-PEI nanoparticles and ADSAM. CD-PEI and PEI nanoparticles used in this study have similar shape, size, and surface charge (Figure 1, right), and SAM and AD-SAM surfaces have similar composition and
hydrophobicity (Table 1). Therefore, these results indicate that CD-PEI nanoparticles are specifically immobilized on the chip surface by cyclodextrin-adamantane inclusion complex formation. To further investigate the interaction between CD-PEI nanoparticles and AD-SAM, 10 mM β-cyclodextrin solutions were introduced to the AD-SAM sensor chips (time ) 5 min; Figure 4, top). Some β-cyclodextrin/AD-SAM interaction was detected (time ) 15 min; Figure 4, top). Addition of CD-PEI nanoparticles resulted in additional interaction with AD-SAM; the immobilized nanoparticles are not removed even after addition of a large excess of free β-cyclodextrin and washing (time ) 75 min; Figure 4, top). As a control, maltoheptaose, a linear oligomer of seven glucose molecules, was used for comparison to cyclodextrin. Addition of 10 mM maltoheptaose solution to the AD-SAM resulted in no observable change in resonant wavelength (time ) 15 min; Figure 4, bottom). These studies again confirm that β-cyclodextrin molecules interact with specificity with AD-SAM. In addition, the multivalent interactions between β-cyclodextrins on single nanoparticles and the AD-SAM significantly increase the binding avidity to the surface such that nanoparticles are not displaced even with competition by β-cyclodextrin solutions at >10 000-fold concentrations. Similar observations have been noted for adamantane-dendrimer molecules associated with cyclodextrin-derivatized SAMs and have been attributed to thermodynamic stability of assemblies.22 The advantages of harnessing multivalency in self-assembly of nanostructures has been well-discussed in a recent review article.40 The high-affinity (40) Mulder, A.; Huskens, J.; Reinhoudt, D. N. Multivalency in supramolecular chemistry and nanofabrication. Org. Biomol. Chem. 2004, 2 (23), 3409-3424.
8484 Langmuir, Vol. 22, No. 20, 2006
Park et al.
Figure 6. Representative AFM images of CD-PEI and PEI nanoparticles incubated with AD-SAM and SAM surfaces.
binding of CD-PEI nanoparticles to AD-SAM is important for biomedical applications where nanoparticles from surfaces might be displaced by biological molecules such as lipids that may form inclusion complexes with β-cyclodextrin. The amount of CD-PEI and PEI nanoparticles immobilized on surfaces was determined by a fluorescence-based assay for DNA content. The plasmid DNA in the nanoparticles can be released by addition of excess heparan sulfate, a polyanionic macromolecule that competitively binds with the cationic polymers and releases plasmid DNA into solution (Figure 5, top).27 The released DNA is then quantified by addition of PicoGreen, a small-molecule dye that fluoresces with maximum emission ∼520 nm when bound to double-stranded DNA. The amount of DNA originally immobilized on the surface is determined by use of a standard curve generated with known concentrations of CD-PEI or PEI nanoparticles treated with heparan sulfate. A high density of CD-PEI nanoparticles was immobilized on AD-SAM surfaces (1289 pg of DNA/mm2 area), whereas pairings with only host molecules (CD-PEI nanoparticles on SAM) or only guest molecules (PEI nanoparticles on ADSAM) resulted in low (
