Novel Strategy for Microsphere-Mediated DNA Transfection

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Novel Strategy for Microsphere-Mediated DNA Transfection Jessica G. Borger,#,† Juan Manuel Cardenas-Maestre,#,‡ Rose Zamoyska,*,† and Rosario M. Sanchez-Martin*,‡,§ †

Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Ashworth Laboratories, King's Buildings, West Mains Road, Edinburgh, EH9 3JT, United Kingdom ‡ School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, United Kingdom

bS Supporting Information ABSTRACT: A new approach for microsphere-mediated delivery of plasmid DNA has been developed and successfully evaluated. Basic molecular biology techniques were used to linearize and functionalize plasmid DNA by aminomodification, enabling efficient conjugation to carboxy-functionalized microspheres. A T cell hybridoma line was successfully transfected as determined by the efficient expression of a biologically relevant YFP fusion protein. Moreover, our data identified microsphere-mediated delivery of plasmid DNA as a noninvasive, nontoxic, and efficient gene delivery method with the potential to be applied to transfection-resistant, nondividing primary cells, including naïve T cells.

T

he development of an efficient carrier system for delivery of plasmid DNA (pDNA) into cells is a key technology for the progress of research in the biological sciences and medicine.1 In the past few years, different technologies and methodologies have been developed in order to efficiently deliver nucleotides at the cellular level.2 Examples include cell penetrating peptides,3,4 cationic lipids,5,6 nanodevices such as biodegradable nanoparticles7 and nanotubes,8 dendrimers,9,10 polymer-mediated delivery including cationic polymers,11 cationic amphiphiles,12 and quantum dots,13 in addition to more invasive techniques such as particle bombardment14 and electroporation.15 Even though all these approaches are currently available, there is still a need to improve transfection efficiency for specific cell lines that are difficult to transfect. We have previously reported that amino-functionalized, cross-linked polystyrene microspheres of highly defined sizes (200 nm to 2 μm) are efficient delivery agents, which can be taken up by a wide range of cell lines including adherent, suspension, primary, and stem cells.16
18 We have achieved the effective microsphere-based delivery of siRNA19 and proteins20 among other biological cargos.21
23 Recently, we reported the ability of palladium-loaded microspheres to carry out intracellular chemical reactions and their potential use to activate drugs and probes inside cells.24 While the mechanism of uptake is not yet established, we have recently found that these microspheres are unlikely to enter cells through an endocytic pathway. The application of chemical inhibitors of endocytosis and extensive colocalization studies by microscopy and gene-expression profiling all argue against an endocytic mechanism. Instead, we have proposed an endocytosis-independent uptake mechanism that results in the uncompartmentalized, r 2011 American Chemical Society

cytoplasmic localization of microspheres and their cargo.25 The fact that these latex microspheres are easy to functionalize with high controllability over the cargo loading, in addition to showing no undesired cytotoxic effect, make them enormously attractive as a novel DNA carrier/delivery system. Here, we describe a novel conjugation approach for microsphere-mediated delivery of pDNA inside cells resulting in expression of the encoded protein. pDNA was linearized and functionalized with 5-(3-aminoallyl)-200 -deoxyuridine 500 triphoshate dUTP (aminoallyl-dUTP) by an established molecular biological approach, to enable its conjugation to microspheres, which were subsequently used to transfect various cell lineages. To carry out this strategy, 200 nm polystyrene microspheres (1) were functionalized as described in Scheme 1 following a standard Fmoc solid-phase protocol, using Oxyma and N,N0 diisopropylcarbodiimide (DIC) as coupling reagents. The carboxy-linker (3,30 -dithiodipropionic acid), which contains a disulfide bond, enabled the efficient attachment of pDNA to microspheres to build construct (3) (Scheme 1). This is an efficient strategy for the delivery of the pDNA from the carrier by incorporation of cleavable bonds to enable controlled molecular release from the particle surface.The introduction of disulfide bonds into carrier molecules is an excellent approach to create an interactive delivery system that exploits the redox gradient between the extra- and intracellular compartments. Examples Received: June 3, 2011 Revised: August 26, 2011 Published: September 07, 2011 1904

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Bioconjugate Chemistry of delivery of bioactive cargos such as nucleic acid or proteins based on the introduction of disulfide bonds into carrier molecules such as chitosan or polymeric nanocapsules have been recently reported.26,27 The efficiency of this strategy relies on the release of biological cargos from nanoparticles following cleavage of cargo
nanoparticle bonds inside biological systems. The stability associated with the disulfide bond linkage ensured that pDNA remained coupled to the microsphere during incubation in culture media. Upon cellular uptake, the nature of the disulfide bond ensured that it was cleaved within the cytosol, releasing the DNA to enable transcription and translation of the encoded Scheme 1. Microsphere Preparation for Conjugation to Linearized pDNAa

a

Reagents and conditions: (i) Fmoc-PEG-OH spacer (5 equiv), Oxyma (5 equiv), DIC (5 equiv), DMF, 2 h, 60 °C; (ii) 20% piperidine in DMF, 3  20 min; (iii) 3,30 -Dithiodipropionic acid (5 equiv), DIC (5 equiv), DIPEA (0.1 equiv), DMF, 2 h, 60 °C.

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protein. Additionally, microspheres were double-pegylated with a Fmoc monoprotected poly(ethylene glycol) spacer (FmocPEG-OH) before being carboxyfunctionalized with a cleavable linker to give rise to microspheres (2) (Scheme 1).28 There were two rationales to double-pegylate the microspheres: first, the addition of these units facilitated transport across the cell membrane by increasing bead biocompatibility, and second, the distance between the DNA and cellular vehicle was increased, which reduced the likelihood of unfavorable interactions. The subsequent conjugation of linearized pDNA with carboxy-functionalized 200 nm polystyrene microspheres (3) (Scheme 1) involved the conjugation of a modified nucleotide aminoallyl-dUTP (4) to DNA (Scheme 2). Initially, pDNA encoding a yellow fluorescent protein (YFP)-tagged protein was linearized at a restriction endonuclease site upstream of the promoter region and amino-functionalized through the incorporation of a modified nucleotide to its 30 -end terminal. Specifically, aminoallyl-dUTP was incorporated into the pDNA by terminal deoxynucleotidyltransferase (TdT), a specialized DNA polymerase which catalyzes the addition of nucleotides to the 30 terminus of a DNA molecule. The incorporation of an amino-reactive group onto the DNA strand enabled the conjugation of the pDNA to the carboxy-functionalized microsphere through formation of an amide bond. The aminoallyl modification enabled downstream reactions with amine-reactive compounds such as activated esters; thus, the aminoallyl-modified pDNA could be conjugated to carboxy-functionalized microspheres (3) in a single step as detailed in Scheme 2. In order to validate beadfection as a novel technology for introduction and expression of DNA into transfection-resistant cell types, we selected immune cells as our model. Unlike other lineages, such as fibroblasts or endothelial cells, which are amenable to transfection by a variety of techniques, naïve T lymphocytes are small, nonproliferating, metabolically inert cells that are readily killed by most standard transfection protocols. T

Scheme 2. Strategy for Conjugation of DNA to Microspheresa

a

Reagents and conditions: (i) TdT (20 units), 10 TdT buffer, CoCl2, 37 °C, 20 min; (ii) 1. EDC, H2O, rt, 4 h; 2. PBS pH 7.4, 18 h, rt. 1905

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Figure 1. F5.BW hybridoma pDNA beadfection and protein expression. (A) Flow cytometric analysis of efficiency of pDNA conjugation to 200 nm microspheres by TOPRO-3 staining. (B) YFP protein expression observed 24 h after incubation: (i) Amaxa electroporation, (ii) beadfection. (C) Flow cytometric analysis of YFP expression in electroporated cells (Amaxa), pDNA-microsphere beadfected cells (Beadfection) and negative controls: untreated cells (No beads) and beads without pDNA conjugated (Naked beads). (D) Confocal microscopy image of a single T cell hybridoma (F5.BW) loaded with pDNA
microsphere conjugate 6 (DIC) expressing PEP-YFP (green). Image taken after 24 h at a 63 magnification, where nuclei are stained with DAPI.

lymphocytes can be activated in vitro by incubation with specific antigen or antibodies directed to the T cell receptor, whereupon they increase in cell size (from approximately 5
7 μM to ∼12 μM) and enter into division. However, murine T lymphocytes, in particular, remain resistant to, and easily killed by, most standard DNA transfection methods. Development of an efficient and noninvasive system for gene delivery into nonproliferating, undifferentiating, or other transfection resistant immune cells would greatly facilitate research into the basic biology of lymphocytes that has been employed by other disciplines for many years. The ability to transfect naïve T lymphocytes remains a challenge that currently is not met by the relatively few transfection technologies available, all of which are associated with high toxicity, low efficiency, and relative lack of reproducibility.29,30 In order to test the beadfection protocol in T lymphocytes, we initially used an immortalized T cell hybridoma line (F5.BW). Given the relatively small size of T lymphocytes, we first evaluated

the optimum size and number of microspheres that gave the most efficient delivery using fluorescein (FITC)-labeled microspheres following a previously described procedure16 (see Supporting Information). After 24 h incubation, microspheres of 200 nm were found to be taken up most readily by the F5.BW hybridoma T cells, as the larger bead sizes were either less efficient (500 nm) or not taken up at all (900 nm) (Figure S1A-B, Supporting Information). Additionally, a linear increase in the efficacy of microsphere uptake was observed, which was proportional to the concentration of microspheres. In addition to microsphere uptake, cell viability was monitored by exclusion of a membrane-impermeable, DNA-intercalating dye, TO-PRO-3 Iodide (TO-PRO-3). Importantly, there was no change in the proportion of cells dying after incubation with the microspheres, although there was evidence of the association of smaller microspheres with dying cells (Figure S1C, Supporting Information). Analysis of the time course of microsphere uptake was carried out 1906

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Bioconjugate Chemistry (Figure S2A-B, Supporting Information), indicated that maximal uptake of 200 nm microspheres was already achieved by 4 h. Mean fluorescent intensity (MFI) was used as a measure of FITC-uptake, and these values remained unchanged following 8 h of incubation and were reduced only slightly at 24 h, which might be due to cell division and subsequent dilution of the numbers of microspheres per cell. Imaging of cells by confocal microscopy confirmed that the microspheres had entered the cell, localizing within the cytoplasm, excluding the possibility that the microspheres had simply adhered to extracellular membrane proteins (Figure S2C, Supporting Information). To validate the use of these microsphere-based conjugates as a novel technology for DNA transfection of immune cells, we addressed whether pDNA could be delivered to F5.BW hybridoma T cells using microspheres as the transfection agent. For this purpose, pDNA encoding a lymphocyte-specific protein, proline-glutamine-serine-threonine-domain enriched phosphatase (PEP), fused at the carboxy-terminus to yellow fluorescent protein (PEP-YFP), was prepared as described in Scheme 1 and conjugated to microspheres as described in Scheme 2. To assess the efficiency of coupling of the pDNA to the microspheres, the DNA intercalator TO-PRO-3 was incubated with the microsphere suspension for 5 min after which the microspheres were washed and analyzed by flow cytometry for the presence of DNA. Figure1A shows that although the naked 200 nm microspheres stained with TO-PRO-3, covalent attachment of pDNA increased the MFI 10-fold, indicative of covalently coupled pDNA.Successful gene expression was achieved upon delivery of conjugate (6) (Scheme 2) to F5.BW hybridoma T cells as determined by the detection of YFP fluorescence by flow cytometry. As shown in Figure1Bii, the majority of the F5.BW hybridoma T cells expressed the PEP-YFP protein 24 h after transfection with pDNA conjugated microspheres (6). Coupling the microspheres with 7.5 μg of DNA rather than 5 μg provided a slight increase in transfection efficiency. As a positive control, F5. BW hybridoma T cells were transfected by the Amaxa (Lonza) electroporation method, currently the most efficient means of transfecting T lymphocytes.31
33 Using the same relative concentration of DNA (5 μg/1  106 cells), F5.BW hybridoma T cells were Amaxa transfected and assessed by flow cytometry for YFP expression 24 h later (Figure1Bi) and 48 h later (Figure S3, Supporting Information). Notably, after 24 h beadfection led to a more uniform expression of PEP-YFP, as seen by a shift of the entire population of transfected F5.BW hybridoma T cells in Figure 1Bii. By comparison, only half as many cells were YFP+ following Amaxa electroporation (Figure1C). However, protein expression decreased dramatically after 48 h in both the Amaxatransfected and the beadfected cells (Figure S3). Endogenous PEP protein has previously been characterized by Western blot to reside wholly within the cytoplasm in T lymphocytes,34 and transfected PEP was shown to localize within the cytoplasm in the nonimmune cell line HeLa.35 Using confocal microscopy, we identified that the transcribed PEP-YFP protein similarly localized within the cytoplasm of F5.BW hybridoma T cells (Figure1D). These results show that a much higher efficiency of transfection is achieved into hybridoma T cells when the pDNA is delivered by beadfection rather than the traditional method of electroporation. The efficient delivery of pDNA and subsequent rapid protein expression in hybridoma T cells led us to address whether naïve T lymphocytes isolated from murine lymph nodes could uptake microspheres. If so, this would identify a novel technique for the

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introduction of DNA into nondividing primary immune cells, which are resistant to more common transfection techniques including lipid carriers and calcium phosphate. Surprisingly, naïve CD8+ T lymphocytes were as efficient in the uptake of FITC-labeled microspheres as the F5.BW hybridoma T cells (Figure S4AB), with a similar dependency on size and concentration of the microspheres. Two important considerations in the transfection of naïve T lymphocytes are whether the DNA delivery protocol leads to either cell death or cellular activation. We confirmed that neither occurred, as there was very little change in the proportion of dead cells (TO-PRO-3+, Figure S4C) and there was no expression on the cell surface of a marker of cellular activation, CD69 (Figure S4D). In contrast, the positive control for activation, which were naïve T lymphocytes stimulated with antibody to the T cell receptor (anti-CD3, Figure S4D) did show expression of CD69. Imaging of the cells by confocal microscopy confirmed that the microspheres had entered the naïve T lymphocytes and localized within the cytoplasm, as no fluorescence was observed within the nucleus (Figure S4E). These results show an efficient internalization of microspheres by naïve T lymphocytes, and consequently, they support the potential of this technology to carry out gene expression studies in this cell line and other resistant primary cells. In conclusion, we have described a novel strategy of transfection, based on the delivery of pDNA by conjugation to microspheres. Following a simple protocol for the linearization and functionalization of the pDNA, this amino-modified DNA was successfully conjugated to 200 nm polystyrene microspheres and transfected into hybridoma T cells leading to successful expression of a biologically relevant protein fused to YFP. In addition, the microspheres were taken up by naive T lymphocytes, a primary cell which is difficult to transfect, with no associated toxicity. These data confirm the development of a novel, noninvasive, efficient, and controlled dose transfection methodology, specifically aimed at DNA delivery into transfection resistant cells that is not matched by currently available technology.

’ ASSOCIATED CONTENT

bS

Supporting Information. Details of synthesis and experimental methods: Experimental procedures for preparation of functionalized microspheres, DNA linearization and aminoallyldUTP incorporation, DNA-microspheres conjugation and biological evaluation. Supplementary Figures: S1 to S4. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Prof. Rose Zamoyska, E-mail: [email protected], Tel: 44-131-651-3686, Fax: 44-131-650-6564. Dr. Rosario Maria Sanchez-Martin, E-mail: [email protected], Tel: +44 (0) 131 650 4713, Fax: +44 (0) 131 650 6453. Present Addresses §

Department of Medicinal and Organic Chemistry, Faculty of Pharmacy, University of Granada, Campus Universitario Cartuja s/n, 18071 Granada, Spain ([email protected]). Author Contributions #

These authors contributed equally to this work.

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’ ACKNOWLEDGMENT This work was supported by the Royal Society and the Medical Research Council (MRC) Ph.D. Fellowship (J.G.B). R.M.S.M would like to thank the Royal Society for a Dorothy Hogdkin Fellowship. J.M.C.M. thanks the School of Chemistry for a Ph.D. Fellowship. ’ REFERENCES (1) Glover, D. J., Lipps, H. J., and Jans, D. A. (2005) Towards safe, non-viral therapeutic gene expression in humans. Nat. Rev. Genet. 6, 299–310. (2) Mintzer, M. A., and Simanek, E. E. (2009) Nonviral vectors for gene delivery. Chem. Rev. 109, 259–302. (3) Bertrand, J. R., Malvy, C., Auguste, T., Toth, G. K., Kiss-Ivankovits, O., Illyes, E., Hollosi, M., Bottka, S., and Laczko, I. A. (2009) Synthesis and studies on cell-penetrating peptides. Bioconjugate Chem. 20, 1307– 1314. (4) Diaz-Mochon, J. J., Bialy, L., Watson, J., Sanchez-Martin, R. M., and Bradley, M. (2005) Synthesis and cellular uptake of cell delivering PNA-peptide conjugates. Chem. Commun. 3316–3318. (5) Martin, B., Sainlos, M., Aissaoui, A., Oudrhiri, N., Hauchecorne, M., Vigneron, J. P., Lehn, J. M., and Lehn, P. (2005) The design of cationic lipids for gene delivery. Curr. Pharm. Res. 11, 375–394. (6) Unciti-Broceta, A., Moggio, L., Dhaliwal, K., Pidgeon, L., Finlayson, K., Haslett, C., and Bradley, M. (2011) Safe and efficient in vitro and in vivo gene delivery: tripodal cationic lipids with programmed biodegradability. J. Mater. Chem. 21, 2154–2158. (7) Blum, J. S., and Saltzman, W. M. (2008) High loading efficiency and tunable release of plasmid DNA encapsulated in submicron particles fabricated from PLGA conjugated with poly-L-lysine. J. Controlled Release 129, 66–72. (8) Singh, R., Pantarotto, D., McCarthy, D., Chaloin, O., Hoebeke, J., Partidos, C. D., Briand, J. P., Prato, M., Bianco, A., and Kostarelos, K. (2005) Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors. J. Am. Chem. Soc. 127, 4388–4396. (9) Diaz-Mochon, J. J., Fara, M. A., Sanchez-Martin, R. M., and Bradley, M. (2008) Peptoid dendrimers—microwave-assisted solidphase synthesis and transfection agent evaluation. Tetrahedron Lett. 49, 923–926. (10) How, S. E., Unciti-Broceta, A., Sanchez-Martin, R. M., and Bradley, M. (2008) Solid-phase synthesis of a lysine-capped bis-dendron with remarkable DNA delivery abilities. Org. Biomol. Chem. 6, 2266– 2269. (11) Peng, L., Liu, M., Xue, Y. N., Huang, S.-W., and Zhuo, R. X. (2009) Transfection and intracellular trafficking characteristics for poly(amidoamine)s with pendant primary amine in the delivery of plasmid DNA to bone marrow stromal cells. Biomaterials 30, 5825– 5833. (12) Zhang, X.- X., Prata, C. A. H., McInstoch, T. J., Barthelemy, P., and Grinstaff, W. (2010) The effect of charge-reversal amphiphile spacer composition on DNA and siRNA delivery. Bioconjugate Chem. 21, 988–993. (13) Zhang, P., and Liu, W. (2010) ZnO QD@PMAA-co-PDMAEMA non viral vector for plasmid DNA delivery and bioimaging. Biomaterials 31, 3087–3094. (14) Uchida, M., Li, X. W., Mertens, P., and Alpar, H. O. (2009) Transfection by particle bombardment: Delivery of plasmid DNA into mammalian cells using gene gun. Biochim. Biophys. Acta 1790, 754–764. (15) Potter, H., and Heller, R. (2010) Transfection by electroporation. In Current Protocols in Molecular Biology, Units 9.3.1
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