Gold Nanoparticle-Mediated Transfection of Mammalian Cells

Bioconjugate Chem. , 2002, 13 (1), pp 3–6 ..... ACS Applied Materials & Interfaces 2009 1 (9), 1980-1987 .... The Journal of Physical Chemistry C 0 ...
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Bioconjugate Chem. 2002, 13, 3−6

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COMMUNICATIONS Gold Nanoparticle-Mediated Transfection of Mammalian Cells Kulmeet K. Sandhu,† Catherine M. McIntosh,† Joseph M. Simard,† Sallie W. Smith,‡ and Vincent M. Rotello*,†

Bioconjugate Chem. 2002.13:3-6. Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 06/24/18. For personal use only.

Department of Chemistry and Department of Veterinary & Animal Science, University of Massachusetts, Amherst, Massachusetts 01003. Received August 16, 2001

Mixed monolayer protected gold clusters (MMPCs) functionalized with quaternary ammonium chains efficiently transfect mammalian cell cultures, as determined through β-galactosidase transfer and activity. The success of these transfection assemblies depended on several variables, including the ratio of DNA to nanoparticle during the incubation period, the number of charged substituents in the monolayer core, and the hydrophobic packing surrounding these amines. Complexes of MMPCs and plasmid DNA formed at w/w ratios of 30 were most effective in promoting transfection of 293T cells in the presence of 10% serum and 100 µM chloroquine. The most efficient nanoparticle studied (MMPC 7) was ∼8-fold more effective than 60 kDa polyethylenimine, a widely used transfection agent.

Gene transfer can be performed using either viral or synthetic gene delivery systems (1-11). While viral vectors are widely used, synthetic transfection systems provide several advantages including their ease of production (7) and the reduced risk of cytotoxicity (7, 12) and immune responses (3, 4, 7, 12, 13). A variety of cationic systems have been studied for complexation and transport of negatively charged DNA, including peptides (2, 7), liposomes containing cationic lipids (14), poly(lysine) (6), polyethylenimine (3), cationic polymers (4, 11), PAMAM dendrimers (1, 5, 10), and more recently, silica nanoparticles (8, 9). Mixed monolayer protected gold clusters (MMPCs) represent a unique platform for transfection because of their ease of fabrication, especially in contrast to the complex synthesis required to produce the highergeneration dendrimer counterparts (15). In addition to simple hydrogen bonding or electrostatic moieties, biological tags capable of targeting the plasmid to a specific cell type are available (7) and can be introduced in postfabrication steps since subsequent modification of the monolayer is also facile. Cationic MMPCs bind very efficiently to DNA; in recent studies we have demonstrated that the interaction between cationic gold nanoparticles and DNA is efficient enough to totally inhibit in vitro DNA transcription by T7 RNA polymerase (16). To explore the utility of MMPCs for transfection, MMPCs featuring a 2 nm gold core, with a total diameter of 6 nm, were fabricated. In comparison with other nanoparticle systems studied (8, 9), we expect that the * Corresponding author: Vincent M. Rotello, Department of Chemistry LGRT 701, 710 N. Pleasant St., University of Massachusetts, Amherst, MA 01003. rotello@ cisco.chem.umass.edu. Phone: (413) 545-2058. Fax: (413) 545-4490. † Department of Chemistry. ‡ Department of Veterinary & Animal Science.

Figure 1. MMPCs used for transfection.

smaller size of the MMPCs should increase the efficiency of both the DNA-MMPC complex internalization and the subsequent release of the plasmid from the cationic MMPC surfaces. For our studies, we fabricated MMPCs modified with quaternary ammonium salts and tested their ability to transfect plasmid DNA (Figure 1).1 The effect of varying cationic coverage was explored by comparing MMPCs 1-5, constructed using differing 1 MMPCs were prepared as previously described in Simard, J., Briggs, C., Boal, A. K., and Rotello, V. M. (2000) Chem. Commun. 1943. Briefly, for 1-5, 50 mg of octanethiol colloid was stirred for two days in THF with 10-100 mg of N,N,Ntrimethyl(11-mercaptoundecyl)ammonium chloride. For 6 and 7, 62 mg of undecanethiol colloid and 50 mg of tetradecanethiol colloid were stirred with 50 mg of ammonium thiol, respectively. After exchange, the MMPCs were purified by mulitple precipitations from dichloromethane. The colloids were characterized using UV, NMR, and TEM methods.

10.1021/bc015545c CCC: $22.00 © 2002 American Chemical Society Published on Web 12/12/2001

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Figure 2. Agarose gel electrophoresis of the MMPCs and DNA. Exterior lanes contain molecular weight markers. The second lane is DNA alone at 9.5 nM; lanes 3-7 contain increasing amounts of MMPC 3 (2.4 µM, 3 µM, 4 µM, 6 µM, and 12 µM, respectively). As the nanoparticle concentration increases, more DNA is retained at the baseline, with complete complexation observed at 12 µM MMPC 3 or at a 790:1 MMPC 3 to DNA mole ratio. Decreasing fluorescence of the DNA fraction at the baseline may be due to absorption of the emitted light by the gold cores or the exclusion of ethidium bromide from the bound and condensed DNA (17).

ratios of octanethiol to ammonium thiol.2 The effect of alkyl chain length on transfection was studied using MMPCs 6 and 7, prepared with undecanethiol and tetradecanethiol, respectively. Preliminary evidence for efficient MMPC-plasmid association was provided by the complete inhibition of electrophoretic mobility of the plasmids upon addition of cationic MMPCs. Gel electrophoresis of pET16B plasmids after incubation with MMPC 3 for 0-30 min showed the presence of only two bands of DNA: one at the baseline and one at the expected molecular weight for a single plasmid (Figure 2).3 The ability of the nanoparticles to completely inhibit the DNA from progressing toward the positive electrode and the lack of any intermediate assemblies indicates either that the complexes formed were completely charge neutralized or that the resultant complexes were too large to enter the gel.4 Preliminary investigations focused on optimization of transfection conditions. Transfection was first compared in different environments: in the presence of both serum and chloroquine (chlq), transfection was improved in comparison to controls, as demonstrated using MMPC 3 (Figure 3a). For all further studies, a medium containing 100 µM chloroquine5 and 10% serum was used.6 To determine the optimal MMPC-plasmid ratio, increasing amounts of MMPC 3 were added to 250 µL of medium containing 5 µg of β-galactiosidase plasmid (Figure 3b).7 Maximum transfection of β-galactosidase reporter gene 2 Cationic coverage of each MMPC was calculated by endgroup analysis using NMR integration. 3 Gels were prepared with 0.6% agarose, run for 60-90 minutes at 80 V, and visualized using ethidium bromide. pET16B plasmids encoding cellular retinoic acid binding protein consist of approximately 6200 base pairs. The molecular weights are composed of linear DNA, whereas the DNA of the pET vector (seen at approximately 4000 base pairs) corresponds to a circular strand. The small band evident in lane 2 at a higher MW is most likely due to incomplete purification of the DNA and is not expected to bias the results observed. 4 Similar DNA:MMPC assemblies were prepared and characterized via methods in reference 16. 5 Cholorquine is believed to disrupt the endosomal vesicle, resulting in increased passage of the transfection vector to the cytosol (1, 2, 9, 11).

Figure 3. Efficiency of transfection as a function of (a) chloroquine (chlq) and serum (FBS) with a 2200:1 MMPC 3:DNA ratio and (b) varying MMPC concentrations.9 Levels of β-galactosidase were determined by comparison to a standard curve using known protein concentrations.

plasmid into 293T cells occurred at a MMPC:plasmid mole ratio of 2200:1. Further increases in MMPC concentration above this value resulted in a slow decrease in transfection efficiency.8 Significantly, the optimal DNA-MMPC ratio for transfection was higher than that required for complete retardation of electrophoretic mobility (vide supra). This is consistent with previous studies that have shown overall net-positively charged assemblies to provide more efficient transfection (9, 18, 19). 6 β-Galactosidase plasmid DNA at 0.02 mg/mL was mixed with MMPC at 5 mg/mL. The complexes were then diluted with DMEM containing 10% FBS and 100 mM chloroquine. The human embryonic kidney cells (293T) were washed once with PBS and incubated with the MMPC/DNA complexes for 3 h at 37 °C in 5% CO2. The cells were washed once with PBS and incubated for 48 h in DMEM + 10% FBS. The enzyme activities were then assayed using a β-galactosidase kit (Promega). The BCA assay (Pierce) was used to determine total protein content. Control experiments performed using naked DNA, nanoparticles alone, and neither DNA nor nanoparticles exhibited no measurable β-galactosidase activity (results not shown). 7 The CMV-β-galactosidase plasmid is composed of approximately 7200 base pairs. 8 Decreasing levels of β-galactosidase expression may be due to low level toxicity of the MMPCs (LD50 ) 1.8 µM (a 2900:1 MMPC:DNA molar ratio) as determined for MMPC 3 using a WST-1 assay). This is ∼165-fold higher than that of PEI (9).

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293T cells, confirming the viability of MMPCs as transfection agents. The efficiency of the transfection process is determined by several parameters, including the ratio of MMPC:DNA used in initial complexation, the surface coverage of quaternary ammonium salts, and the length of the uncharged thiol chain. Further studies of the role of monolayer structure and functionality are currently underway and will be reported in due course. ACKNOWLEDGMENT

Figure 4. Transfection of β-galactosidase mediated by formation of various MMPC-DNA complexes at 2200:1 nanoparticle/ DNA ratio.

We thank Lila Gierasch and Jennifer Habink for the donation of cells containing the pET16B plasmid. We also thank Barbara A. Osborne for the use of her tissue culture facility. This research was supported by the National Science Foundation (DMR-9809365 for MRSEC instrumentation) and the National Institutes of Health (GM-59249 and GM-62998). V.M.R. acknowledges the Alfred P. Sloan Foundation, the Camille and Henry Dreyfus Foundation, and is a Cottrell scholar of Research Corporation. C.M.M. acknowledges support from the National Institutes of Health Chemistry-Biology Interface Training Grant GM-08515. LITERATURE CITED

Figure 5. Transfection with MMPCs 2, 6, 7 (2200:1 nanoparticle/DNA ratio) and PEI (60 kDa).

We next explored the effect of the ratio of quaternary ammonium endgroups to hydrophobic chains (Figure 4). All of the gold nanoparticles were able to promote transfection in the presence of chloroquine and serum, with MMPC 3 proving to be the most effective of the octanethiol substituted particles. This suggests that either the charge/particle ratio or the hydrophobicity of the MMPCs is an important determinant of transfection efficiency. The effect of the chain length of the monolayer was determined by testing the transfection efficiency of MMPCs 6 and 7 (Figure 5). In these studies, increasing transfection efficiency was observed with increasing chain length. All three nanoparticles were more efficient than polyethylenimine (PEI), with MMPC 7 being approximately eight times as effective as PEI.10 In summary, we have demonstrated the efficient transfection of plasmids encoding β-galactosidase into 9 MMPC concentrations were determined by weight (based on an average molecular weight of 60000 for the nanoparticle, see ref 20) and DNA concentrations measured by UV-vis. 10 PEI experiments performed as described in ref 9.

(1) Luo, D., and Saltzman, W. M. (2000) Enhancement of transfection by physical concentration of DNA at the cell surface. Nat. Biotechnol. 18, 893-895. (2) Singh, D., Bisland, S. K., Kawamura, K., and Gariepy, J. (1999) Peptide-based intracellular shuttle able to facilitate gene transfer in mammalian cells. Bioconjugate Chem. 10, 745-754. (3) Coll, J. L., Chollet, P., Brambilla, E., Desplanques, D., Behr, J. P., and Favrot, M. (1999) In vivo delivery to tumors of DNA complexed with linear polyethylenimine. Hum. Gene Ther. 10, 1659-1666. (4) Lynn, D. M., and Langer, R. (2000) Degradable poly(betaamino esters): Synthesis, characterization, and self-assembly with plasmid DNA. J. Am. Chem. Soc. 122, 10761-10768. (5) Tang, M. X., Redemann, C. T., and Szoka, F. C. (1996) In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem. 7, 703-714. (6) Perales, J. C., Ferkol, T., Molas, M., and Hanson, R. W. (1994) An Evaluation of Receptor-Mediated Gene-Transfer Using Synthetic DNA-Ligand Complexes. Eur. J. Biochem. 226, 255-266. (7) Morris, M. C., Chaloin, L., Heitz, F., and Divita, G. (2000) Translocating peptides and proteins and their use for gene delivery. Curr. Opin. Biotechnol. 11, 461-466. (8) Kneuer, C., Sameti, M., Haltner, E. G., Schiestel, T., Schirra, H., Schmidt, H., and Lehr, C. M. (2000) Silica nanoparticles modified with aminosilanes as carriers for plasmid DNA. Int. J. Pharm. 196, 257-261. (9) Kneuer, C., Sameti, M., Bakowsky, U., Schiestel, T., Schirra, H., Schmidt, H., and Lehr, C. M. (2000) A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro. Bioconjugate Chem. 11, 926-932. (10) Bielinska, A. U., Chen, C. L., Johnson, J., and Baker, J. R. (1999) DNA complexing with polyamidoamine dendrimers: Implications for transfection. Bioconjugate Chem. 10, 843-850. (11) Arigita, C., Zuidam, N. J., Crommelin, D. J. A., and Hennink, W. E. (1999) Association and dissociation characteristics of polymer/DNA complexes used for gene delivery. Pharm. Res. 16, 1534-1541. (12) Luo, D., and Saltzman, W. M. (2000) Synthetic DNA delivery systems. Nat. Biotechnol. 18, 33-37. (13) Tripathy, S. K., Black, H. B., Goldwasser, E., and Leiden, J. M. (1996) Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of

6 Bioconjugate Chem., Vol. 13, No. 1, 2002 replication-defective adenovirus vectors. Nat. Med. 5, 545550. (14) Colin, M., Harbottle, R. P., Knight, A., Kornprobst, M., Cooper, R. G., Miller, A. D., Trugnan, G., Capeau, J., Coutelle, C., and Brahimi-Horn, M. C. (1998) Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells. Gene Ther. 11, 1488-1498. (15) Tomalia, D. A., Naylor, A. N., and Goddard, W. A., III. (1990) Starburst dendrimers: Molecular level control of size, shape, surface, chemistry and topology. Angew. Chem., Int. Ed. Engl. 29, 138-175. (16) McIntosh, C. M., Esposito, E. A., Boal, A. K., Simard, J. M., Martin, C. T., and Rotello, V. R. (2001) Inhibition of DNA Transcription Using Cationic Mixed Monolayer Protected Gold Clusters. J. Am. Chem. Soc. 123, 7626-7629. (17) Gershon, H., Ghirlando, R., Guttman, S. B., Minsky, A. (1993) Mode of Formation and Structural Features of DNA Cationic Liposome Complexes Used for Transfection. Biochemistry 32, 7143-7151.

Sandhu et al. (18) Truong-Le, V. L., Walsh, S. M., Schweibert, E., Mao, H. Q., Guggino, W. B., August, J. T., and Leong, K. W. (1999) Gene transfer by DNA-gelatin nanospheres. Arch. Biochem. Biophys. 361, 47-56. (19) Wolfert, M. A., Schacht, E. H., Toncheva, V., Ulbrich, K., Nazarova, O., and Seymour, L. W. (1996) Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block copolymers. Hum. Gene Ther. 7, 21232133. (20) Hostetler, M. J., Wingate, J. E., Zhong, C.-J., Harris, J. E., Vachet, R. W., Clark, M. R., Londono, J. D., Green, S. J., Stokes, J. J., Wignall, G. D., Glish, G. L., Porter, M. D., Evans, N. D., Murray, R. W. (1998) Alkanethiolate Gold Cluster Molecules with Core Diameters from 1.5 to 5.2 nm: Core and Monolayer Properties as a Function of Core Size. Langmuir 14, 17-30.

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