Mesoporous silica nanoparticles deliver DNA and chemicals into plants FRANC¸OIS TORNEY1, BRIAN G. TREWYN2, VICTOR S.-Y. LIN2* AND KAN WANG1* 1
Center for Plant Transformation, Plant Science Institute and Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA Department of Chemistry, US DOE Ames Laboratory, Iowa State University, Ames, Iowa 50011, USA *e-mail: [email protected]
; [email protected]
Published online: 29 April 2007; doi:10.1038/nnano.2007.108
Surface-functionalized silica nanoparticles can deliver DNA1–8 and drugs9–15 into animal cells and tissues. However, their use in plants is limited by the cell wall present in plant cells. Here we show a honeycomb mesoporous silica nanoparticle (MSN) system with 3-nm pores that can transport DNA and chemicals into isolated plant cells and intact leaves. We loaded the MSN with the gene and its chemical inducer and capped the ends with gold nanoparticles to keep the molecules from leaching out. Uncapping the gold nanoparticles released the chemicals and triggered gene expression in the plants under controlledrelease conditions. Further developments such as pore enlargement and multifunctionalization of these MSNs may offer new possibilities in target-specific delivery of proteins, nucleotides and chemicals in plant biotechnology. The unique structural features of organically functionalized MSNs, such as their chemically and thermally stable mesoporous structures, large surface areas (.800 m2 g21), tunable pore sizes (2–10 nm in diameter) and well-defined surface properties, have made them ideal for hosting guest molecules of various sizes, shapes and functionalities. In many non-porous particle-based delivery systems for plant cells and tissues, the molecules of interest are limited to nucleic acids, which are usually adsorbed on the exterior particle surface of the carriers. For example, in the widely used ‘gene gun’ system, DNA-coated gold microparticles are used as bullets for bombardment of plant cells and tissues to achieve gene transfer in these cell-wall-containing organisms. Although such systems are suitable for many applications, it is currently difficult to co-deliver anything other than nucleic acids. Microinjection is a method that can be used for delivery of multiple biogenic species including DNA. However, this method is not suitable for plant transformation owing to its limitation in the materials that can be targeted and its extremely low efficiency16. In the case of the MSN system, the drugs and imaging agents are loaded in the mesopores and encapsulated with covalently bound caps that physically block the drugs from leaching out (Fig. 1a). Molecules entrapped inside the pores are released by the introduction of uncapping triggers (chemicals that cleave the bonds attaching the caps to the MSN). We have demonstrated the feasibility of this design by using a disulphide-reducing antioxidant, dithiothreitol (DTT), as the gate-opening trigger to release biogenic molecules inside mammalian cells14. To investigate how this capped MSN system would interact with various plant cells, we synthesized a series of MSNs with different surface functional groups/caps. We first investigated the use of nature nanotechnology | VOL 2 | MAY 2007 | www.nature.com/naturenanotechnology
MSN on protoplasts (plant cells with the cell wall removed), which are often used as model systems in physiological and cellular process studies17. Compared with animal systems, plant cell endocytosis is less understood and the tools for studying the mechanisms are limited to membrane-impermeable dyes18. In our experimental conditions 26+7% of tobacco mesophyll protoplasts underwent endocytosis using Lucifer Yellow dye (see Supplementary Information, Fig. S1). Protoplasts incubated with Type-I MSN (Fig. 1a) did not take up the nanoparticles (Fig. 2a). However, under the same condition, Type-II MSN (that is, Type-I MSN functionalized with triethylene glycol, TEG) was successfully internalized in 7+3% of the cells examined (Fig. 2b) and remained in endocytotic vesicles in the cytoplasm for the entire experimental duration (72 h). This demonstrates that the particle surface properties play a crucial role in plant cell endocytosis. Compared with animal cells, where endocytosis is an efficient process, the amount of MSN per plant cell appeared low. The size of the endocytotic vesicles ranged between 0.2 and 3 mm (diameter), which represents between 1 and 15 MSNs. The number of vesicles per cell was highly variable, ranging from 1 to .20. This is the first example of isolated plant cell endocytosis of TEGcoated nanoparticles. Because no toxicity to plant cells was observed (see Supplementary Information, Fig. S2), the MSN system can serve as a new and versatile tool for plant endocytosis and cell biology studies. To prove that MSNs can function as DNA delivery agents for plant cells, we used a plasmid containing a green fluorescent protein (GFP) gene under the control of a constitutive promoter (see Supplementary Information, Fig. S3a). The optimal coating ratio for DNA/Type-II MSNs was 1/10 (w/w). With this ratio, or less, DNA formed a stable complex with Type-II MSNs, because no free DNA was observed in solution after 2 h of incubation (Fig. 3). Type-II MSN-bound DNAs were not digested by restriction enzymes (see Supplementary Information, Fig. S4). Transient GFP expression could be observed 36 h after protoplasts were incubated with DNA-coated Type-II MSN (Fig. 4a). In all GFP-expressing cells examined ( 7% endocytosis frequency), Type-II MSNs could also be detected inside the cells (Fig. 4a). Standard polyethylene glycol (PEG)-mediated protoplast transformation can achieve 40 –90% transient transformation efficiency when using 1 – 2 mg of DNA per 106 cells (ref. 19). Here, transient transformation of 7% of the cells could be achieved when 106 cells were incubated with 1,000 times less DNA, that is, 1 mg DNA (coated on 10 mg Type-II MSNs). Our results indicate 295
= Fluorescein Gold
= -oestradiol Type-IV
Freed plasmid Type-IV Gold Gold
Type-IV Cell membrane
Figure 1 Mesoporous silica nanoparticles for plant cell internalization. a, Schematic representation of a series of surface-functionalized mesoporous silica nanoparticles (MSNs) for intracellular controlled release of genes and chemicals in plant cells. Linker-MSN, precursor MSN for Type-III and Type-IV; Type-I, fluorescein-labelled MSN; Type-II, fluorescein-labelled, TEG-coated MSN; Type-III, fluorescein-filled MSN capped by gold nanoparticles; Type-IV, b-oestradiol loaded MSN capped by gold nanoparticles. b,c, Transmission electron micrographs of Type-II (b) and Type-III (c) MSNs (scale bar: 100 nm). Arrows indicate gold nanoparticles. 296
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LETTERS Red channel
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DNA/MSN ratio ( g/ g) 1/5
Type-II MSN only
that the DNA-coated Type-II MSN can serve as an efficient delivery system for protoplasts and make the DNA accessible to transcription machinery, leading to the transgene expression. Because intact plant tissues are desired targets for plant genetic engineering, we tested whether we could introduce MSNs into plants with the gene gun system. Attempts to bombard Type-I and Type-II MSNs did not lead to any successful transformation and no MSNs were observed inside the cells, possibly because of the low density of these silica-based particles. To overcome this problem, we synthesized a Type-III MSN, where the mesopores are capped by surface-functionalized gold nanoparticles (10–15 nm in size, Fig. 1a,c), which not only serve as a biocompatible capping agent20, but also add weight to each individual MSN to increase the density of the resulting complex material. GFP-expressing foci can be visualized on tobacco cotyledons bombarded with DNA-coated Type-III MSNs (Fig. 4b,c). On average, 32+11 GFP fluorescent foci were visible per bombarded cotyledon. The use of commercially available 0.6-mm gold particles produced an average of 73+24 GFP-expressing foci per cotyledon. These results show that the gold nanoparticle-capped Type-III MSNs can be used for delivering DNA into intact plant cells and tissues by using the gene gun system. Furthermore, in comparison with the gold particle-based DNA delivery systems, where the DNAs are coated on the surface of solid gold particles, the mesoporous structure of the MSNs (surface area 900 m2 g21) (ref. 14) offers the possibility of loading a large quantity of biogenic moieties, including chemicals that are membrane impermeable or incompatible with cell growth media, inside the pores and delivering them along with DNA to the targeted cells.
Circular plasmid DNA
Figure 2 Confocal imaging of MSN uptake by tobacco mesophyll protoplasts. Protoplasts incubated with a,b,c, Type-I MSNs (single focal plane images) and d,e,f, Type-II MSNs (three-dimensional reconstruction images). No uptake of Type-I MSNs was observed but Type-II MSNs were internalized. Both MSNs are functionalized with fluorescein and visible in green (thick arrows). Autofluorescing chloroplasts in the protoplasts are in red (thin arrows).
DNA bound to MSN Free DNA
Figure 3 Identifying the optimal DNA coating on Type-II MSNs. MSNs were incubated with purified circular plasmid DNA (11 Kb) in water at various DNA:MSN ratios for 2 h at 23 8 C. The entire incubation mix was then electrophorized in 1% agarose gel and DNA bands were stained with ethidium bromide. Free DNA (thin arrow) migrates in the gel whereas bound DNA (thick arrow) remains in the wells. The optimal DNA coating for a Type-II MSN is 1:10 (w/w).
We also used DNA-coated Type-III MSNs to bombard maize immature embryos. GFP-expressing callus sectors from a proliferating callus culture grown on non-selective medium were visible ten days after the bombardment (Fig. 4d,e). Although the gold nanoparticles may be aggregated on the MSN surface, the results suggested that the DNA could still form stable electrostatic complexes on the surface of a Type-III MSN, and that this genetransfer system could be used to generate both transient and stable transgenic plant materials. The most advantageous feature of the MSN technology is its potential to deliver different biogenic species simultaneously to 297
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Figure 4 Gene expression in plant cells incubated with MSNs. a, Threedimensional reconstruction of two protoplasts. The top cell contains an endocytosed MSN (thick arrow) expressing GFP and the bottom cell lacks MSN. Auto-fluorescing chloroplasts are in red (thin arrows) (scale bar: 10 mm). Tobacco leaf bombarded with DNA-coated Type-III MSN in b, bright field and c, ultraviolet light/GFP filter (scale bar: 0.2 mm). Callus developing from immature maize embryos bombarded with DNA-coated Type-III MSN under d, bright field and e, ultraviolet light/GFP filter (scale bar: 1 mm).
the target sites and release the encapsulated chemicals in a controlled fashion. To test whether the controlled release used for animal cells6,21,22 could also be applicable to plant cells, we generated transgenic tobacco containing an inducible promoter controlled GFP gene (see Supplementary Information, Fig. S3b). We chose two transgenic events: one with low (event B) and one with higher (event G) integration pattern complexity (Fig. 5a –d; see also Supplementary Information, Methods). The expression of GFP in plants can only be observed when the chemical inducer b-oestradiol is present23. Fourteen-day-old transgenic plantlets were bombarded with Type-IV MSNs (Fig. 1b), which were filled with equal amounts of b-oestradiol (see Supplementary Information, Methods), and their pores capped with gold nanoparticles by means of the disulphide chemical linker14. The release of b-oestradiol is triggered by DTT, which is a commonly used chemical in the media for enhancing plant transformation frequency24. An average of 62.5+6.1 and 51.2+12.4 fluorescent foci were counted for the transgenic events B and G, respectively, germinated on DTT-containing medium (Fig. 5e,g). Fewer than 298
five foci were detected on control plants germinated on DTT-free medium and bombarded by Type-IV MSNs, and on plants bombarded with FITC-filled Type-III MSNs, regardless of the media types. The detection of sporadic fluorescent foci on transgenic plants in the control experiments is likely due to either low-level promoter leakage of this b-oestradiol inducible system or stress responses caused by the bombardment treatment. No DTT-induced stresses were detectable from plants. This result indicates that a gene present in the plant genome could be activated by chemicals delivered into the cell, leading subsequently to controlled released, in planta, by using this MSN system. Finally, to demonstrate that the MSN system can simultaneously deliver both the gene and the chemical that triggers gene expression, we performed experiments in which non-transgenic plants were bombarded with b-oestradiol-loaded, gold nanoparticle-capped MSNs (Type-IV, Fig. 1a,c) coated with the inducible GFP marker gene (see Supplementary Information, Fig. 3b). An average of 35.6+12.8 fluorescent foci could be detected on plantlets germinated in DTT-containing medium (Fig. 5h,i). Only low levels of background fluorescent foci (,5 foci, on average) were detected in control treatments, including plants germinated on DTT-free medium and plants bombarded with FITC-filled Type-III MSNs coated with an inducible GFP marker gene. These data indicate that we can deliver DNA molecules carrying a marker gene and a chemical that is needed for transgene expression into plant cells simultaneously and release the encapsulated chemical in a controlled manner to trigger the expression of co-delivered transgene in the cell. Current applications of nanotechnology to biology have been mainly focused on animal science and medical research. Here, we have demonstrated that their versatility can also be applied to plant science research to aid further investigation of plant genomics and gene function as well as improvement of crop species.
METHODS MESOPOROUS SILICA NANOPARTICLES
Fluorescein-doped MSNs with approximate diameter of 100–200 nm (FITCMSN, Type-I MSN) were synthesized as described previously22. For the synthesis of Type-II MSNs, the surface of Type-I MSNs were modified by covalently attaching an organic ligand, TEG. The synthesis of the TEG ligand was achieved via a modified literature procedure25. Specifically, we first synthesized 1-[2-(2-bromo-ethoxy)-ethoxy]-2-methoxy-ethane by following the reported procedure25 directly. The isolated molecule was then attached to 3-aminopropyltrimethoxysilane by refluxing in ethanol overnight. The crude product was directly grafted to the as-synthesized FITC-MSN (still containing the surfactant template). Once grafted, the surfactant template was removed as described in our previous report13. To construct the DNA delivery systems, Linker-MSN material (MSNs with amine terminal organic linkers reducible by DTT) with an average pore size of 2.3 nm was synthesized as described14. To load the analytes (fluorescein or b-oestradiol), the Linker-MSN (100 mg) was suspended in a buffer or buffer/organic solution of the chosen analyte (2.6 1024 M fluorescein or 5.0 1022 M b-oestradiol) and stirred at room temperature for 24 h. Acid functional gold nanoparticles were synthesized by modifying the method described by Brust and colleagues26. Specifically, we substituted the organic thiol ligand used by Brust (p-mercaptophenol) with 11-mercaptoundecanoic acid (see Supplementary Information, Methods). For capping, these acid gold nanoparticles were suspended in the same analyte solutions and, along with amide coupling agent (80 mg), were stirred at room temperature with the Linker MSN for 24 h. Following centrifugation and washings, gold-capped fluorescein or b-oestradiol loaded MSNs were isolated. The amount of fluorescein loaded (22 mmoles g21) was calculated by measuring the difference in the initial loading solution and the fluorescein that was physisorbed and removed by the washings. nature nanotechnology | VOL 2 | MAY 2007 | www.nature.com/naturenanotechnology
LETTERS B NI
100.0 DTT No DTT
Fluorescent foci per cotyledon
80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 MSN type MSN content Event
Figure 5 GFP expression in tobacco plants induced by MSN-mediated delivery of b-oestradiol. Transgenic cotyledons (event B) grown with (I) and without (NI) b-oestradiol under a, bright field b, ultraviolet light/GFP filter, c, merged (scale bar: 2 mm). d, Southern blot analysis of transgenic tobacco plants for events B and G. (2): non-transgenic plant; (þ): pER8-GFP plasmid. e, Bright field and f, ultraviolet light/GFP filter (scale bar: 0.1 mm) image of cells expressing GFP. g, Fluorescent foci per transgenic cotyledon grown with (grey bar) or without (black bar) DTT after bombardment with MSNs. h, Bright field and i, ultraviolet light/GFP filter (scale bar: 0.5 mm) images of non-transgenic plants in DTT-medium and bombarded by DNA-coated Type-IV MSNs.
Protoplasts were isolated from 6- to 8-week-old tobacco plants (Nicotiana tabacum cv Petite Havana) aseptically grown on Murashige and Skoog media without plant growth hormones (MS) according to a published protocol27. For tobacco plantlet bombardment experiments, 25 surfacesterilized seeds were arranged per plate (5 mm apart) in the centre of a 100 25 mm Petri dish containing MS media and germinated aseptically for 14 days (23 8C, 18 h light). For culture conditions of transgenic plants used in controlled-release experiments, surface-sterilized seeds of each transgenic event were germinated on Y-segmented Petri dishes. Each third of a segmented Petri dish contained a different media: (1) MS media, (2) MS þ 1 mM DTT and (3) MS þ 50 mM b-oestradiol. In the plate centre nature nanotechnology | VOL 2 | MAY 2007 | www.nature.com/naturenanotechnology
part of each third, 10 seeds were placed in each segment (30 seeds per plate) and allowed to germinate for 14 days. Maize immature zygotic embryos were isolated from genotype Hi II ears 12 days after pollination28. ENDOCYTOSIS EXPERIMENTS
Tobacco protoplasts were mixed with MSNs in W5 media27 using 106 cells for 10 mg of MSN. The mix was incubated overnight in 3 ml of W5 in BD Falcon six-well flat-bottom plates. To remove non-internalized MSN (except for Fig. 2a), the mixtures were overlaid on K4 medium27 and centrifuged at 100 g for 10 min at room temperature. The protoplasts at the interphase were collected and evaluated. 299
LETTERS DNA/MSN COATING AND BOMBARDMENT
To coat DNA onto Type-II MSNs for endocytosis experiments, 1 mg of purified plasmid DNA was incubated with 10 mg MSN in 50 ml water for 2 h at 23 8C. The MSNs were washed three times with W5 media prior to incubation with protoplasts. Bio-Rad Biolistic PDS-1000/He particle delivery system was used for MSN delivery into plant cells. To coat DNA onto gold-capped MSNs, we used the standard protocol for coating gold particles for the biolistic gun29 with modifications. To prevent the agglomeration, a quick freeze-drying method of the gun macrocarrier was developed. Once the DNA/MSN mixture was in ethanol, the tube was vortexed constantly. Immediately after the DNA/MSN mixture was loaded onto the macrocarrier, it was immersed in liquid nitrogen. The frozen macrocarriers were then freeze-dried using a Labconco Freezone 2.5 for 2 h before being used for bombardment. The bombardment parameters for the tobacco plantlets were 650 p.s.i. rupture disk, 10-cm gap distance and 10-cm target distance. A sterile 150-mm mesh was used between the macrocarrier and the target tissue. Bombardment of maize immature embryos was performed using the laboratory standard procedure28. After bombardment, the embryos were placed on N6-30 medium28 without selection for ten days to avoid callus autofluorescence interfering with the GFP expression evaluation. IN PLANTA CONTROLLED RELEASED
Plants were germinated and grown in Y-segmented Petri dishes as described in the ‘Plant Material’ section. For each transgenic event, nine Petri dishes with a total of 270 plantlets (90 plants per media treatment) were used. Plants were grown for 48 h after bombardment before being subjected to evaluation. Fluorescent foci were viewed under an Olympus dissecting scope (see Supplementary Information, Methods). Transient and stable transgenic analyses are also described in the Supplementary Methods.
Received 23 November 2006; accepted 27 March 2007; published 29 April 2007. References 1. Bharali, D. J. et al. Organically modified silica nanoparticles: A nonviral vector for in vivo gene delivery and expression in the brain. Proc. Natl Acad. Sci. USA 102, 11539–11544 (2005). 2. He, X. et al. A novel DNA-enrichment technology based on amino-modified functionalized silica nanoparticles. J. Disper. Sci. Technol. 24, 633 –640 (2003). 3. Kneuer, C. et al. A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro. Bioconjugate Chem. 11, 926 –932 (2000). 4. Luo, D., Han, E., Belcheva, N. & Saltzman, W. M. A self-assembled, modular DNA delivery system mediated by silica nanoparticles. J. Control Release 95, 333 –341 (2004). 5. Luo, D. & Saltzman, W. M. Enhancement of transfection by physical concentration of DNA at the cell surface. Nature Biotechnol. 18, 893 –895 (2000). 6. Radu, D. R. et al. A polyamidoamine dendrimer-capped mesoporous silica nanosphere-based gene transfection reagent. J. Am. Chem. Soc. 126, 13216– 13217 (2004). 7. Roy, I. et al. Optical tracking of organically modified silica nanoparticles as DNA carriers: A nonviral, nanomedicine approach for gene delivery. Proc. Natl Acad. Sci. USA 102, 279– 284 (2005). 8. Sameti, M. et al. Stabilisation by freeze-drying of cationically modified silica nanoparticles for gene delivery. Int. J. Pharm. 266, 51 – 60 (2003). 9. Giri, S., Trewyn, B. G., Stellmaker, M. P. & Lin, V. S.-Y. Stimuli-responsive controlled-release delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles. Angew. Chem. Int. Edn Engl. 44, 5038–5044 (2005). 10. Gruenhagen, J. A., Lai, C. Y., Radu, D. R., Lin, V. S.-Y. & Yeung, E. S. Real-time imaging of tunable adenosine 5-triphosphate release from an MCM-41-type mesoporous silica nanosphere-based delivery system. Appl. Spectrosc. 59, 424 –431 (2005). 11. Hirsch, L. R. et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc. Natl Acad. Sci. USA 100, 13549– 13554 (2003).
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Acknowledgements We thank N.-H. Chua for providing pER8-GFP. Special thanks go to M. Carter for confocal microscope assistance, B. Frame and L. Moeller for discussions, and the Plant Transformation Facility personnel for providing maize immature embryos and maize callus media. The authors thank the Plant Science Institute at Iowa State University for financial support. V.S.-Y.L. thanks the U.S. NSF (CHE-0239570), the US DOE and the Office of Basic Energy Sciences (W-7405-Eng-82) for financial support for the synthesis and characterization of the MSN materials. Correspondence regarding plant transformation should be addressed to K.W. Correspondence regarding nanoparticles and requests for nanoparticle materials should be addressed to V.S.-Y.L. Supplementary information accompanies this paper on www.nature.com/naturenanotechnology.
Author contributions F.T. and K.W. conceived and designed the plant transformation experiments. F.T. performed the plant transformation experiments. V.S.-Y.L. and B.G.T. conceived and designed the surface functionalized mesoporous silica nanoparticle systems for the controlled release of DNAs and chemicals. B.G.T. performed experiments on the synthesis and characterization of the capped mesoporous silica nanoparticle materials. All authors discussed the results and participated in the writing of the manuscript.
Competing financial interests The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/
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