Letter pubs.acs.org/ac
MicroRNA Conjugated Gold Nanoparticles and Cell Transfection Elizabeth Crew, Sharaara Rahman, Asma Razzak-Jaffar, Derrick Mott, Martha Kamundi, Gang Yu,† Nuri Tchah, Jehwan Lee, Michael Bellavia, and Chaun-Jian Zhong* Department of Chemistry, State University of New York at Binghamton, Binghamton, New York, 13902, United States S Supporting Information *
ABSTRACT: While the importance of microRNAs (miRNAs) in cancer treatment or manipulation of genetic expression has been increasingly recognized for developing miRNA-based therapies, the controlled delivery of miRNAs into specific cells constitutes a challenging task. This report describes preliminary findings from an investigation of the conjugation of gold nanoparticles with miRNAs (miRNA− AuNPs) and their cell transfection. The immobilization of miRNAs on the AuNPs was detected, and the surface stability was substantiated by gel electrophoresis assessment of the highly charged characteristics of miRNA−AuNPs and their surface-exchange inactivity with a highly charged surfactant. The miRNA− AuNPs were tested in cell transfection using multiple myeloma cells, demonstrating efficient knockdown in the functional luciferase assay. The findings have important implications for understanding the mechanistic details of cell transfection involving miRNA-conjugated nanoparticles as biosensing or targeting probes.
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Scheme 1. Illustration (Not to Scale) of the Preparation of miRNA−AuNP Conjugates for Delivering miRNAs to Cellsa
here has been increasing interest in delivering small strands of RNA, e.g., microRNA (miRNA), into cells for genetic manipulation or cellular marking.1−19 While significant associations of miRNA with mRNA (mRNA) have been identified in relation to cancer treatment,4 such as the resistance of multiple myeloma (MM) to glucocorticoid treatment,11 the development of methods for effective delivery of miRNAs into appropriate cells without significant cytotoxicity remains challenging. A recent successful example involved the delivery of siRNA (small interfering RNA) conjugated gold nanoparticles into cells and efficient knockdown of target genes without significant cytotoxicity.20 The use of gold nanoparticles as a delivery vehicle exploits their unique optical properties,22 low cytotoxicity,20,21 and enhanced lifespan in the bloodstream.23 Through the labeling of nanoparticles with RNA, certain genetic sequences can be targeted for cancer treatment or repression of certain genes.24 Different methods have recently been reported for analyzing miRNAs, including electrochemical,25 optical,26 fluorescence,27 capillary electrophoresis,28 and spectroscopic29−31 methods. The delivery of miRNA into cells has also been studied using nanoparticles as carriers7,8 and other nanoscale delivery systems.9,10 However, evidence for the immobilization of miRNAs on gold nanoparticles followed by testing cell transfection has yet to be demonstrated. In this study, we investigated the conjugation of miRNAs to gold nanoparticles and its cell transfection (Scheme 1). Note that this strategy was successfully exploited for siRNA delivery in which gold nanoparticles protected the siRNA from RNases, showing effective delivery of the siRNA into cells for gene knockdown.15−19 Both siRNA and miRNA exhibit gene silencing; however, siRNA primarily inhibits translation of mRNA, whereas miRNA primarily cleaves mRNA.32 © 2011 American Chemical Society
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miRNA is labeled with fluorescent dyes (e.g., Cy3 or Cy5 (see Supporting Information Scheme S2-A)).
The importance of miRNA in the treatment of cancer and for the manipulation of genetic expression has been demonstrated recently by Gunaratne, Rosen, and co-workers.11−13 Certain miRNAs (e.g., miR-130b) were found to express differently in glucocorticoid-sensitive versus glucocorticoid-resistant MM.1 cell lines. The overexpression of miR-130b in the MM.1S cell line decreases the expression of a glucocorticoid receptor protein (GR-α), inhibiting glucocorticoid-induced apoptosis of Received: October 17, 2011 Accepted: December 4, 2011 Published: December 8, 2011 26
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cells and causing resistance to glucocorticoids.11 These studies constitute an inspiration for the exploration of gold nanoparticles as carriers of miRNAs in cell transfection. The immobilization of thiol-functionalized miRNAs on gold nanoparticles was characterized by surface-enhanced Raman scattering (SERS) and gel electrophoresis analyses, where SERS detects both miRNAs29,30 and dye-labels30,31 on the nanoparticle surfaces whereas gel electrophoresis analysis35 provides information for assessing the binding and stability of miRNAs on the nanoparticles under conditions of surface ligand exchange. Cell transfection and functional luciferase assays provide further information for ascertaining the delivery of miRNAs into cells.
between the spectra before and after the conjugation with miRNAs, we note however that the detailed assignments of these bands are not complete due to the complication of the weak signals and the overlapping of the bands from Cy5 and miRNA and possible differences in their enhancements. We also note that these SERS bands were often very narrow and weak (Supporting Information Figure S1−I) in comparison with other similar systems,29−31 though less-narrow bands were also observed (Supporting Information Figure S1−II). We believe that the very small quantity of miRNAs per particle (∼15 miRNAs) could have contributed to the weak signals. A further investigation with different quantities of miRNAs per particle is needed to substantiate the spectroscopic characteristics. The assessment of the conjugation and stability of the nanoparticles was performed using agarose gel electrophoresis, testing the highly charged nature of the miRNAs on the AuNPs. Both negative and positive control experiments were performed, including unmodified AuNPs and those treated with bis(p-sulfonatophenyl)phenylphosphine (BP, a negatively charged surfactant often used for increasing the surface charge35). As shown in Figure 1, the unmodified particles
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EXPERIMENTAL SECTION The miRNA oligonucleotide sequences included a sense strand (miR-130b, i.e., 5′-/Phos/CAG UGC AAU GAU GAA AGG GCA UAC/iSp18//iSp18//3ThioMC3-D/-3′) and an antisense strand (miR-130b*, 5′-ACU CUU UCC CUG UUG CAC UAC GCA U-3′), which is labeled with Cy3 or Cy5 at the 5′ end. A multiple myeloma cell line (MM.1S) was used in the cell transfection studies. The surface modification of citrate-capped gold nanoparticles of 13 nm diameter (Au NPs) with miRNAs followed a procedure similar to that reported for modifying AuNPs with siRNA.15 The amount of miRNA added to the solution was sufficient for 10 to 25 miRNAs per nanoparticle. Cell transfection was performed following a standard protocol.11 Multiple myeloma cells were incubated with nanoparticle solutions, and nanoparticle uptake was analyzed under a fluorescent microscope. Analysis of expression of target genes was performed using a functional luciferase assay utilizing GR3′UTR Luciferase Reporter.11 The experimental details are given in Supporting Information.
Figure 1. Photo showing the agarose gel electrophoresis result. Lane 1: citrate-capped AuNPs; Lane 2: citrate-capped AuNPs after treating with BP, i.e., BP-capped AuNPs; Lane 3: miRNA-conjugated AuNPs; and Lane 4: miRNA-conjugated AuNPs after treating with BP.
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RESULTS AND DISCUSSION Conjugation of miRNA to Gold Nanoparticles. The citrate-capped nanoparticles (AuNPs) were first treated with diethyl pyrocarbonate followed by autoclaving treatment. The miRNA duplexes were added to the post-treatment AuNPs, followed by addition of oligoethylene glycol thiol. UV−visible spectroscopic measurements were performed to monitor the change of the nanoparticle solution (see Supporting Information Figure S1). A slight shift in the plasmonic resonance peak position was observed, which can be explained by the changes in the capping and surface ionic characteristics due to the presence of miRNAs on AuNPs. Little change in the size and shape of the nanoparticles was observed, as evidenced by TEM micrographs taken of the AuNPs before and after conjugation with miRNA (not shown). The immobilization of the Cy5 labeled miRNA on the AuNPs was examined using SERS, which exploits the surface plasmonic coupling of the conjugated AuNPs to produce hot spots.33−35 For a sample obtained after conjugation of AuNPs with miRNA and separation by centrifugation, we compared the bands (see Supporting Information Figure S2) with those known for miRNA29 and Cy5.31 In the literature, it is reported that bands at 600, 794, 1306, and 1631 cm−1 are due to miRNA,29 whereas bands at 1594, 1500, 1271, and 1200 cm−1 correspond to ν(CN)stretch, ν(C−C)ring, ν(CC)ring, and ν(C−N)stretch modes for Cy5.31 In our data, bands are observed at around 1590 and 1360 cm−1. While there is a sharp contrast
basically stayed in the well of the gel due to the lack of sufficient surface charges (Lane 1), whereas the BP-capped particles traveled the farthest in the gel as a result of the high level of charges on the particles (Lane 2). In contrast to citrate-capped AuNPs (Lane 1), the miRNA-conjugated AuNPs traveled to a certain distance in the gel (Lane 3), which is indeed consistent with the negative charges of miRNAs on the nanoparticles. When the miRNA-conjugated particles were treated with BP, the gel distance remained basically unchanged (Lane 4), indicating the absence of surface ligand exchange reaction between miRNAs on AuNPs and BPs in the solution. The absence of such surface exchange reactivity provides an important piece of evidence supporting the stability of the miRNA-conjugated AuNPs. Cell Transfection of miRNA-Conjugated Gold Nanoparticles. Samples of the conjugation of gold nanoparticles with miRNAs (miRNA−AuNPs) were tested in cell transfection using a multiple myeloma cell line model (MM.1S). Confocal/fluorescent microscopic measurement was performed to determine the presence of the fluorescent-tagged miRNA− AuNPs in the cells. MM.1S cells were treated with the miRNA−AuNP conjugates following a standard cell transfection procedure,11 where the nanoparticles were taken into the cells by normal endocytotic pathways. Figure 2 shows a representative set of images for the cell transfection. Images 27
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Figure 2. Confocal/fluorescent composite microscopic images of the MM.1S cells 48 h after transfection was initiated with Cy5−labeled miRNA− AuNPs (A, with two different magnifications) and Cy3-labeled miRNA−AuNPs (B). The cells were exposed to a 2 nM nanoparticle solution.
were taken of both a cellular uptake control and the miRNA− AuNPs. The clear uptake of the conjugated nanoparticles was evidenced by the presence of Cy5−miRNA−AuNPs (Figure 2A) and Cy3−miRNA−AuNPs (Figure 2B) in the MM.1S cells. Small quantities of nanoparticles can be seen in nearly all of the cells, with a few cells containing large numbers of nanoparticles, which were found to be necrotic. The cells maintain a similar appearance before and after the cell transfection, as demonstrated by the unchanged outlines of the cells and the clear contrast due to fluorescence from the entrance of the dye-labeled miRNA−AuNPs in Figure 3.
Figure 4. Plots showing the results of the functional luciferase assays for Cy5 labeled miRNA−AuNPs (black bars), Cy3 labeled miRNA− AuNPs (red bars), and a mimic system (inset chart).
miRNAs, which corresponds to an effective miRNA concentration of ∼6 μM in the 10 mL solution or ∼350 miRNA per nanoparticle (see Supporting Information). In the luciferase knockdown, the miRNA−AuNP solution contained a final miRNA concentration of 0.25 μM or ∼15 miRNAs per nanoparticle. The surface coverage was apparently smaller than that reported for siRNA conjugated AuNPs (∼33 siRNAs per particle15) where 3 nM siRNA−AuNPs were used (∼100 nM of siRNA) for a kinetic study in luciferase knockdown and a double stranded siRNA (100 nM) was used as a positive control.15 On the basis of a comparison of the knockdown efficiencies between siRNA−AuNPs (∼20% at 48 h15) and miRNA−AuNPs (∼40% at 48 h), it appeared that the miRNA−AuNPs were somewhat more efficient at a much lower concentration. A higher percentage (∼75%) was reported for siRNA−AuNPs for a longer period of time (96 h).15
Figure 3. Microscopic images of the MM.1S cells prior to cell transfection (A) and after cell transfection with the Cy5-labeled miRNA−AuNPs (B).
Functional luciferase assays were performed to quantitatively determine if the miRNA delivered into the cells by the AuNPs was capable of reducing luciferase generated by a GR3′UTR linked reporter. Both positive and negative controls were performed using miRNA mimics without AuNPs and AuNPs without miRNA. Samples were treated with Cy3- or Cy5tagged miRNA−AuNPs or by cotransfecting miR-130b mimics as a positive control. Figure 4 shows the results of the assays. While little biological effect was observed at low concentrations, the increase of the concentration to 2 nM Cy5−miRNA− AuNPs led to an observable reduction in gene expression. A similar result was observed for the Cy3−miRNA−AuNPs. The reduction in gene expression was found to scale with the concentration of miRNAs. The nanoparticle concentrations of 0.0, 0.5, and 2.0 nM correspond to a miRNA concentration of 0.0, 7.5, and 30 nM, respectively. For a typical 10 mL solution of 16 nM AuNPs, a full monolayer coverage of miRNA would require 4 × 1016
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CONCLUSION In conclusion, the successful conjugation of miRNAs to gold nanoparticles with a relatively high stability has been demonstrated by a simple procedure. The resulting miRNA− AuNPs were shown to exhibit an effective transfection in multiple myeloma cells. It is remarkable that a 4% coverage of miRNAs on the nanoparticles could produce such a knockdown efficiency in the functional luciferase assay. While the determination of the functionalization of the gold nanoparticles 28
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with different dyes and further studies using high miRNA surface coverages are yet to be performed to determine the concentrations of miRNA−AuNPs needed for a high-efficiency knockdown, these findings have implications for the design of miRNA-conjugated gold nanoparticles as effective biosensing and targeting probes, which is part of our further investigation.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text and complete references for refs 2, 4, and 5. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected]. Present Address † College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan, China.
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ACKNOWLEDGMENTS This work is supported by the National Science Foundation (CHE 0848701). Dr. P.H. Gunaratne of Baylor College of Medicine and Mr. M.A. Tessel of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University are gratefully acknowledged for the helpful discussion on delivery of miRNAs and the cell transfection measurement.
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