Microprecision Delivery of Oligonucleotides in a 3D Tissue Model and

Article pubs.acs.org/molecularpharmaceutics

Microprecision Delivery of Oligonucleotides in a 3D Tissue Model and Its Characterization Using Optical Imaging Zhen Luo,†,‡ Ting Ye,†,‡ Yunzhe Ma,§ Harvinder Singh Gill,*,§ and N. Nitin*,† †

Department of Biological and Agricultural Engineering, University of California, Davis, Davis, California 95616, United States Department of Chemical Engineering, Texas Tech University, Lubbock, Texas 79409, United States

§

S Supporting Information *

ABSTRACT: Despite significant potential of oligonucleotides (ONs) for therapeutic and diagnostic applications, rapid and widespread intracellular delivery of ONs in cells situated in tissues such as skin, head and neck cavity, and eye has not been achieved. This study was aimed at evaluating the synergistic combination of microneedle (MN) arrays and biochemical approaches for localized intratissue delivery of oligonucleotides in living cells in 3D tissue models. This synergistic combination was based on the ability of MNs to precisely deliver ONs into tissues to achieve widespread distribution, and the ability of biochemical agents (streptolysin O (SLO) and cholesterol conjugation to ONs) to enhance intracellular ON delivery. The results of this study demonstrate that ON probes were uniformly coated on microneedle arrays and were efficiently released from the microneedle surface upon insertion in tissue phantoms. Co-insertion of microneedles coated with ONs and SLO into 3D tissue models resulted in delivery of ONs into both the cytoplasm and nucleus of cells. Within a short incubation time (35 min), ONs were observed both laterally and along the depth of a 3D tissue up to a distance of 500 μm from the microneedle insertion point. Similar widespread intratissue distribution of ONs was achieved upon delivery of ON−cholesterol conjugates. Uniformity of ON delivery in tissues improved with longer incubation times (24 h) postinsertion. Using cholesterolconjugated ONs, delivery of ON probes was limited to the cytoplasm of cells within a tissue. Finally, delivery of cholesterolconjugated anti-GFP ON resulted in reduction of GFP expression in HeLa cells. In summary, the results of this study provide a novel approach for efficient intracellular delivery of ONs in tissues. KEYWORDS: antisense, cholesterol, imaging, microneedle, oligonucleotide delivery, silencing, streptolysin O, tissue

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To address the first challenge, i.e., limited penetration and distribution of ONs in target tissues, both systemic and localized delivery methods have been studied. For systemic circulation, the key challenges for the delivery of ONs include poor stability of ONs in serum, cytotoxic effects due to nontargeted accumulation of ONs in organs such as liver and kidneys, and nonspecific activation of the immune system.4−8 In addition, molecular targeting ligands selected for the specific delivery of ON probes must have high specificity for the target cells. While systemic in vivo delivery is attractive for certain target tissues such as liver, heart, kidney, and metastasized tumor cells, there are various therapeutic and diagnostic applications in which the localized delivery of ONs can be more effective.9−13 Examples of these applications include the silencing of target genes in skin, eye, lungs, cervix, oral cavity (such as localized oral tumors), and a range of diagnostic applications such as imaging of gene expression in clinically abnormal tissues.

INTRODUCTION

Intracellular delivery of oligonucleotides (ONs) in 3D tissues is challenging. There is an unmet need to develop a methodology to efficiently deliver ONs to individual cells within a tissue. Delivery of ONs in tissues has significant potential for therapeutic applications based on silencing of gene expression using antisense and siRNA molecules. In addition, delivery of ON probes can also enable novel diagnostic and theranostic applications. For example, molecular beacons upon hybridization with target RNA molecules can generate a specific fluorescent signal enabling detection of diseased cells.1,2 On the other hand, ON probes modified with photosensitizer molecules that become activated upon hybridization of ON probes with target RNA molecules3 can induce photodamage in cells that express particular RNA molecules to simultaneously provide a therapeutic and a diagnostic action. Two major barriers limit successful intracellular delivery of ONs in an intact 3D tissue. The first challenge is related to the current inability to achieve widespread distribution of ONs in the tissue such that all cells in the tissue can be exposed to a sufficient therapeutic dose of ONs. The second challenge is related to the poor intracellular delivery of ONs. © XXXX American Chemical Society

Received: December 18, 2012 Revised: May 2, 2013 Accepted: May 28, 2013

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dx.doi.org/10.1021/mp300717f | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Table 1a sequence no. ON_1 ON_1_chol Anti_GFP a

oligonucleotide sequence and modifications

purpose

5′-CAA ACT CCA GGT TTT CTT TCC representative ON sequence tagged with Cy3 to allow fluorescence imaging of intratissue C/3Cy3Sp/-3′ and intracellular distribution of ONs 5′-/5Cy3/CAA ACT CCA GGT TTT ON_1 sequence conjugated with cholesterol to determine the ability of cholesterol to CTT TCC C/3CholTEG/-3′ increase ON transport into cells; Cy3 label allows visualization G*A*G CTG CAC GCT GCC G*T*C/ designed to hybridize with GFP-RNA to suppress its expression into GFP 3CholTEG/3′

mol wt 7241.0 7858.9 6296.8

Cy3: Cyanine 3 dye with peak excitation at 550 nm and peak emission at 570 nm. CholTEG: Cholesterol linked with a triethylene glycol spacer.

combine (i) microneedles as devices to precisely deliver ONs into tissues with extensive intratissue distribution of ON probes with (ii) biochemical agents to enhance penetration of the widely dispersed ONs into cells. This synergism was designed to simultaneously address both the challenges associated with poor ON delivery into cells situated in a tissue environment. Microneedles, which are sharp microstructures arranged in an array format and an easy-to-use patch, provide a minimally invasive approach for efficient delivery of ONs into the tissue over a large area; while the biochemical agent then enhances the intracellular delivery of ONs from the extracellular matrix of the tissue. In this study, two biochemical approaches to enhance intracellular delivery of ONs were evaluated. In the first approach, streptolysin O (SLO) coated on microneedles was delivered to tissues to induce nanoscale pores in cell membranes for enhancing intracellular delivery of ONs. SLO has been extensively used in cell culture to deliver ONs43,44 for silencing and imaging gene expression. In the second biochemical approach cholesterol was conjugated to ONs. Conjugation of cholesterol to ONs has previously demonstrated significant improvement in intracellular delivery of ONs in 2D cell culture models.45−47 Epithelial-derived cancer cells were cultured in a collagen gel to create a 3D tissue structure. Previous studies have shown that structural and optical properties48−50 and diffusion properties of macromolecules in this 3D tissue model are in agreement with measurements in tumor tissues.51,52 By varying delivery methods (different combinations of microneedle and biochemical agents) and incubation time, intratissue and intracellular distributions of ONs in the 3D tissue models were characterized using optical imaging. Functional activity of ON probes was characterized based on antisense-mediated suppression of the green fluorescent protein (GFP) expression in the 3D tissue model. To the best of our knowledge, this is the first study that has used microneedles to address the difficulty in achieving widespread delivery in lateral and vertical dimensions of ONs in tissues and to evaluate the synergistic combination of microneedles and biochemical approaches for achieving efficient intracellular delivery of ONs.

Localized delivery is an attractive option for the selected tissues because of ease of application and potentially lower dose requirements as compared to systemic delivery. Prior studies have evaluated passive topical diffusion, iontophoresis, and direct injection of ONs using hypodermic needles.9,12,14−17 Results have shown that passive topical diffusion of ONs in epithelial tissues is significantly limited due to the relatively large molecular weight and net negative charge of ONs.18−20 To overcome these limitations of topical delivery, direct injection using hypodermic needles has been used. Although direct injection is effective for the delivery of a large dosage of ONs, it has limited ability to target epithelial cells that are located within 300−500 μm from the mucosal surface. Furthermore, the uniformity of delivery of ONs over a large cross-section of a tissue is significantly limited using hypodermic needles.21 Although iontophoresis and electrophoresis are attractive approaches, their clinical application has yet to be realized. Potential side effects from the long-term use of electrical fields on living tissue are also an unknown variable.22,23 Recently micrometer-sized sharp structures called microneedles have been developed to precisely target the skin for localized, painless, and minimally invasive delivery of drugs and vaccines.24,25 Among the different approaches that exist to deliver drugs using microneedles, the coated microneedle approach is attractive because of its simple implementation and potential thermal stability offered because the drug is in a solid dry state on the microneedle.26 A large number of compounds have been coated on microneedles including desmopressin, a short peptide,27 ovalbumin,28 hepatitis C DNA vaccine,29 and microparticles.28 In the majority of the applications, microneedles have been used either for vaccination or for delivery of drugs for systemic action. The inherent capability of microneedles when arranged as an array to uniformly deliver drugs with widespread distribution locally in tissues has not yet been exploited. Accordingly, we propose to use coated microneedles for the first time to deliver ONs into tissues with the goal of achieving widespread distribution of ONs locally in the tissue. To address the second challenge, i.e., poor ON penetration into the cell, many nonviral approaches based on physical and biochemical approaches have been developed. Examples of the physical nonviral delivery approach include direct microinjection and electroporation.30−34 Examples of the biochemical approaches for nonviral delivery of ONs include the design and development of polyplexes (complexation of charged DNA/ RNA backbones with oppositely charged oligomers and polymers) and conjugation of ONs with peptides, such as Tat peptide and small molecules (folic acid, cholesterol).15,35−39 Despite these developments, significant challenges in the delivery of ONs to the target tissue remain, especially in vivo.4,40−42 The objective of this study was to synergistically

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MATERIALS AND METHODS Materials. The human cervical carcinoma cell line, HeLa, was a gift from Professor Glenn M. Young (University of California, Davis). The HeLa/GFP cell line was purchased from Cell Biolabs, Inc. (San Diego, CA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), MEM nonessential amino acids (NEAA), trypsin, and trypan blue were obtained from Fisher Scientific (Pittsburgh, PA). Penicillin−streptomycin, Tris (2-carboxyethyl)phosphine hydrochloride (TCEP), and streptolysin O (SLO) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Type I B



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removal of the coated ON probe from the microneedle surface. A standard curve based on a serial dilution of the fluorescently labeled ON probes was generated to determine the concentration of the released ONs. Cell Culture and 3D Tissue Models. The human cervical carcinoma cell line, HeLa, was maintained in a culture medium consisting of DMEM (Fisher Scientific, Pittsburgh, PA) supplemented with 10% FBS (Fisher Scientific, Pittsburgh, PA) and 100 mg/L penicillin (Sigma, St. Louis, MO). HeLa cells (4 × 104 cells/mL) were seeded into culture flasks, grown in a humidified atmosphere of 5% CO2−95% air at 37 °C, and subcultured with 0.05% trypsin (Fisher Scientific, Pittsburgh, PA). The HeLa/GFP cell line was maintained using the same protocol as the HeLa cell line with the only modification that 0.1 mM MEM nonessential amino acids (NEAA) was added to the culture medium. GFP expressing HeLa cells were used for evaluating efficacy of the anti-GFP ONs in suppressing GFP expression in 3D tissue models. 3D tissue models were prepared using HeLa or HeLa/GFP cells embedded in a collagen matrix. Type I collagen (Roche USA) was dissolved in 0.2% acetic acid to 3 mg/mL. To prepare a 3D tissue model with high cell density, a suspension of HeLa or HeLa/GFP cells was spun down and resuspended in a small volume of DMEM media (Invitrogen, Carlsbad, CA) containing 5% FBS to reach a cell density of 1 × 108 cells/mL. The collagen solution and cell suspensions were then mixed in a 2 to 1 ratio (by volume) to obtain a final collagen concentration of 2 mg/mL. 1 M NaOH was gradually added into the mixture to achieve a final pH of 7.4. Then the suspension was transferred into a 24 mm Transwell with a 3.0 μm pore polycarbonate membrane at the bottom (Corning Incorporated, Corning, NY). These Transwells were then placed in a 24-well plate with 500 μL of DMEM containing 5% FBS. The collagen−cell matrix was allowed to gel at 37 °C for 30 min. After forming the tissue model, 40 μL of cell culture medium was added to the top of the model. Tissue models were incubated at 37 °C for approximately 48 h. This incubation promotes the formation of a highly dense 3D tissue model (total volume of approximately 120 μL). Assessment of Viability of Cells after the Insertion of Microneedles in 3D Tissue Models. To evaluate the impact of microneedle insertion on cell viability in a tissue model, a trypan blue exclusion assay was performed. Trypan blue exclusion assay is a well-established method to determine integrity of cell membranes and viability of cells: damaged, nonviable cells get stained blue while intact, and viable cells do not take up the blue stain. For this assay, microneedles were inserted in 3D tissue models and incubated for 5 min. After removal of microneedles, the tissue model was transversely sliced and the sections were stained using 0.4% trypan blue. Transverse sections from the negative control tissue models (without insertion of microneedles) were also prepared and stained with trypan blue. Sections from both the negative control and the microneedle-treated tissue model were imaged using an inverted microscope (IX71 Olympus Inc., Center Valley, PA). Results of the control and treated tissue were compared to evaluate the impact of microneedle insertion on cell viability. Evaluation of Intracellular Distribution of ONs in 3D Tissue Models. Delivery of ONs Using the Combination of Microneedles and SLO. SLO was first activated by adding 500 mM TCEP to 200 U/mL of SLO for 30 min at 37 °C. Excessive TCEP was then removed from the solution using a

collagen from rat tail was obtained from Roche, Inc. (South San Francisco, CA). ON probes were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Design and Fabrication of Microneedle Array. A linear array containing five microneedles at an intramicroneedle spacing of 1.5 mm28,53 was fabricated from 50 μm thick stainless steel (304) sheets using a wet etch process. Each microneedle in the array measured 700 μm in length and 200 μm in width, and tapered to a sharp tip. Design of ON probes. The design of ON probes used in this study is shown in Table 1. All ON probes were synthesized using solid phase synthesis and purified using HPLC (Integrated DNA Technologies, Inc. (Coralville, IA)). The sequence of Anti_GFP ON was selected on the basis of the results of a previously published study.54 The internucleotide linkage between the last two nucleotides on both the 5′ and 3′ termini of the Anti_GFP ON was modified to a phosphorothioate linkage. Nucleotides with the modified internucleotide linkage are indicated with an asterisk in Table 1. Coating of ONs on Microneedles. To develop a uniform coating of ONs with a precise control over the length of the microneedles being coated, an automated X−Y-axis microprecision dip-coating process was used. Microneedles were mounted on an automated X−Y computer-controlled stage. The coating solution was housed in an orifice into which the microneedles were dipped through motion control of the X−Y stage. The coating solution was composed of FDA-approved excipients,53,55−57 including 1% (w/v) carboxymethylcellulose sodium salt (low viscosity, USP grade, CarboMer, San Diego, CA, USA) and 0.5% (w/v) Lutrol F-68 NF (BASF, Mt. Olive, NJ, USA). ONs at a concentration of 200 μM were added to the coating solution to achieve a final coating concentration of 100 μM. Microneedles were consecutively dipped into the coating solution to build up coatings on microneedle surfaces. Uniformity of Coated ONs on Microneedles. The coating uniformity of ONs on microneedles was characterized using both widefield and high resolution fluorescence imaging. For the widefield imaging, both white light and fluorescence images of the coated microneedle array were acquired using a widefield imaging system (Maestro 2, CRI, Woburn, MA). Fluorescence images were obtained using an excitation filter (BP 540) and a bandpass emission filter (BP 570−600). The camera exposure time for the widefield imaging measurements was 100 ms. For the high resolution imaging, an inverted fluorescence microscope (IX71 Olympus Inc., Center Valley, PA) was used. The excitation and emission filters for the high resolution microscopy were 560/15 nm and 590−630 nm respectively. The camera exposure time for the high resolution imaging measurements of a microneedle sample was 10 ms. Efficiency of Release of Coated ONs in Collagen Gels. To demonstrate rapid and efficient release of the coated ONs in collagen gels, microneedles coated with ON probes were inserted into a collagen gel (2 mg/mL collagen) for 5 min. Release efficiency of the coated ONs was characterized based on quantitative fluorescence imaging and spectroscopy. Using fluorescence imaging, the mean fluorescence intensity of ON probes on the microneedle surface before and after insertion in a collagen gel was quantified. The amount of the ON on the surface of the microneedles before and after insertion in a collagen gel was also measured using fluorescence spectroscopy. To measure the amount of ON, the microneedle samples (both before and after insertion) were incubated with 1× PBS at 50 °C for 30 min. This incubation process resulted in complete C



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Figure 1. Characterization of ON-coated microneedle arrays. (a) Widefield images (white light and fluorescence) of a microneedle array to evaluate uniformity of coating of fluorescently labeled ONs on microneedle arrays. (b) High resolution images of an individual microneedle in the array to evaluate uniformity of the coated ONs on microneedles. To illustrate the release of fluorescently labeled ONs upon insertion in a collagen gel, the high resolution images were acquired before and after insertion of a microneedle array in a collagen gel. (c) Changes in the mean fluorescence intensity of coated ONs on individual microneedles before and after insertion in a collagen gel. Scale bar represents 200 μm.

spin desalting column (Zeba Spin Desalting Columns, 7K MWCO, Thermo Scientific, Rockford, IL). The activated SLO was next coated on microneedles using the same coating solution and procedure as used for the coating of ONs. First, ON-coated microneedles were inserted into a 3D tissue model for 5 min and removed. Next, SLO-coated microneedles were inserted into the tissue model for 5 min. The concentration of ONs and SLO coated on each microneedle array was approximately 0.136 nmol of fluorescently labeled ON_1 and 1.0−1.5 units of SLO, respectively. The control tissue models were treated with just the ON-coated microneedle array. The tissue models were incubated for 35 min at 37 °C post removal of the microneedle arrays. This short incubation time period was selected on the basis of the experimental conditions used in the 2D cell culture assays for intracellular delivery of ON probes using SLO. After incubation, tissue models were fixed with 4% paraformaldehyde for 30 min, stained with 0.1 mM DAPI (4′,6-diamidino-2phenylindole, a DNA binding dye) for 10 min, and imaged using confocal microscopy. Delivery of Cholesterol-Conjugated ONs. For the delivery of cholesterol-conjugated ONs, microneedles coated with ON_1_chol were inserted into a 3D tissue model followed by a 5 min incubation at 37 °C. After removal of microneedles, tissue models were incubated for either 35 min or 24 h at 37 °C. After incubation, tissue models were fixed, stained with DAPI using the same procedure as outlined above, and imaged using confocal microscopy. Imaging Distribution of ONs in 3D Tissue Models. Distribution of ONs was characterized as a function of radial

distance from the microneedle insertion points and along the depth of insertion of microneedles in the tissue. Using intact tissue models microneedle insertion points can be easily detected, and the distribution of ONs as a function of radial distance from the microneedle insertion points was mapped with a mechanically controlled stage. To map variation in fluorescence intensity along the tissue depth, transverse sections (cross-section) of 3D tissue models were prepared and imaged using confocal microscopy. Transverse sections were prepared by embedding tissue models in 5% agar gel (w/ w %) and transversely sectioning the gels using an oscillating tissue slicer (EMS 5000, Electron Microscopy Sciences Inc., Hatfield, PA). Confocal fluorescence images (Zeiss LSM 510) were acquired using a combination of laser excitations at 543 nm and at 780 nm on a multiphoton Ti-sapphire mode locked laser. Fluorescence emission from the Cy3-labeled ONs and the nuclear staining dye (DAPI) was collected using band-pass emission filters of 560−600 nm and 400−440 nm, respectively. For image acquisition, the multichannel module of the Zeiss LSM confocal imaging software was used. Images were acquired using both 25× (NA-0.8) and 40× (oil, NA-1.43) objectives. Efficacy of Antisense-Mediated Silencing of Target Gene Expression in 3D Tissue Models. To evaluate the functional activity of ONs in 3D tissue models, efficacy of an antisense ON in silencing target gene expression was evaluated. GFP expression was selected as a target for this functional assay due to ease of visualization. Microneedle arrays coated with anti-GFP (same concentration as described for the ON_1) were inserted into tissue models. After removal of the microneedle array, tissue models were incubated for 36 h at D



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Figure 2. Delivery of ONs in a 3D tissue model using a synergistic combination of microneedle arrays and streptolysin O (SLO). (a) Characterizes the radial distribution of ONs in an intact 3D tissue model as a function of distance from the microneedle insertion point (white arrow). Distances are calculated from the microneedle insertion point in a tissue phantom to the center of the adjacent regions. (b) Characterizes the axial distribution of ONs in a transverse cross-section of a tissue. (c) Variation in the mean fluorescence intensity as a function of distance from the top surface of the transverse cross-section of a tissue model. Scale bar represents 50 μm.

37 °C. Control tissue models were treated with microneedles without any oligonucleotide coating. After incubation, tissue models were imaged using confocal microscopy. GFP signal was detected using a 488 nm laser excitation and a band-pass emission filter (510−550 nm). For each sample, multiple images were taken at different locations in a tissue model. Image Analysis. NIH Image J software was used for the quantitative analysis of the imaging data. To quantify the mean fluorescence intensity, the RGB images were converted to the gray scale images and the mean fluorescence intensity over a selected region of interest was quantified. The regions of interest for quantification of the mean fluorescence intensity were determined using the white light reflectance images that outlined the structure of the tissues and their sections. For statistical analysis, the mean fluorescence intensity measurements were made using multiple images (typically 5 images) from three independent repeat experiments. To characterize the intracellular distribution of ONs using the SLO and the cholesterol-conjugated ONs, the fluorescent line scans were generated to quantify the intracellular variation in fluorescence intensity of the ON probes and their spatial correlation with the distribution of the nuclear staining dye.

base structure. The imaging results further show uniform fluorescence intensity on individual microneedles of the array. ON coating uniformity was also evaluated using high resolution imaging. The high resolution imaging measurement shows that the fluorescent signal intensity was relatively uniform across the length of an individual microneedle (Figure 1b) within an array. Overall, these results demonstrate uniform coating of ONs on the microneedle array using the FDA approved excipients. Concentration of the fluorescently labeled ONs per microneedle array was quantified using fluorescence spectroscopy. Based on the fluorescence spectroscopy measurements, each microneedle array was coated with approximately 0.136 ± 0.007 nmol of the fluorescently labeled ON. Microneedle-Coated ONs Were Efficiently Released in Collagen Gels. Figures 1b and 1c show the representative high resolution imaging micrographs of microneedles before and after insertion in a collagen gel, respectively. Release efficiency of the coated ONs from the microneedles upon insertion in a collagen gel was quantified on the basis of changes in the mean fluorescence intensity and amount of the coated ONs on the microneedle arrays. Figure 1d shows more than 90% decrease in the mean fluorescence intensity after insertion of the ONcoated microneedles in a collagen gel. The amount of ON coated on microneedles before and after insertion was also measured using fluorescence spectroscopy measurements as described in Materials and Methods. Results of the fluorescence spectroscopy measurements show that over 90% (preinsertion = 0.136 ± 0.007 nmol of ON per array, postinsertion = 0.0138

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RESULTS ONs Can Be Uniformly Coated on Microneedles. Figure 1a shows that the microneedle array was uniformly coated with fluorescently labeled ON. Importantly, the coatings were only localized to microneedles without contaminating the E



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Figure 3. Delivery of ONs in a 3D tissue model using a synergistic combination of microneedle arrays and cholesterol-conjugated ONs. (a) Characterizes the radial distribution of ONs in an intact 3D tissue model as a function of distance from the microneedle insertion point (white arrow). Distances are calculated from the microneedle insertion point in a tissue phantom to the center of the adjacent regions. (b) Characterizes the axial distribution of ONs in a transverse cross-section of a tissue model. (c) Variation in the mean fluorescence intensity as a function of distance from the top surface of the transverse cross-section. Scale bar: 50 μm.

significant decrease in the mean fluorescence intensity was observed. Figure 2b shows the axial (depth) distribution of ONs in a transverse section of a tissue model. The results show that, by using microneedles, ONs were delivered across the entire thickness of the tissue model (∼500 μm). Figure 2c shows variation in the mean fluorescence intensity as a function of distance along the microneedle insertion direction. The results show that the relative fluorescence intensity near the insertion point is higher as compared to the distal portion of the tissue model. This result indicates that the ONs coated on the microneedle are rapidly released and dispersed upon insertion of the microneedles in tissue models. Imaging measurements (Figure S1 in the Supporting Information) of a control tissue phantom show no significant autofluorescence with the same imaging conditions as in Figure 2. Imaging measurements in Figures 2a and 2b also demonstrate a significant spatial overlap of the nuclear staining dye, DAPI (blue color) and the fluorescently labeled ONs (red). This overlap between the DNA binding dye and the ON probes indicates efficient intracellular delivery of ONs in individual cells within a 3D tissue model by use of SLO. To demonstrate significance of SLO in promoting intracellular delivery of ONs, control 3D tissue models were treated just with ON-coated microneedles without any treatment with SLO-coated microneedles. After a short period of incubation (∼35 min), the control tissue models were imaged using

± 0.0005 nmol of ON per array) of the coated ONs were released from the microneedle array within 5 min of incubation in collagen gels. These results demonstrate that the coated ONs can be efficiently released from the microneedle arrays in a collagen gel. These results are consistent with previous studies58 which have demonstrated similar efficiency in delivery of coated biomolecules using microneedles in the skin tissue. Synergistic Combination between Microneedles and Streptolysin O for Intracellular Delivery of ONs in 3D Tissue Models. To evaluate synergy between microneedles and SLO in promoting intracellular delivery of ONs, 3D tissue models were treated with microneedles coated with ONs and SLO, respectively. Using confocal microscopy, intratissue distribution of ONs along the radial and axial directions with respect to the microneedle insertion direction was measured. The radial distribution of ONs in a tissue model was measured using intact 3D tissue models. Intratissue distribution of ONs along the axial coordinates was measured using a transverse section of a tissue model as described in Materials and Methods. Figure 2a shows the radial (lateral) intratissue distribution of ONs with respect to the microneedle insertion points in an intact tissue model. Imaging results show that by using the microneedle array ONs were efficiently delivered up to a distance of 300−500 μm from the microneedle insertion point within 35 min of incubation. After an approximate radial distance of 500 μm from the microneedle insertion point, a F



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Figure 4. Comparison of subcellular localization of ONs delivered using SLO and cholesterol-conjugated ONs in a 3D tissue model. (a) Imaging measurements to compare subcellular localization of ONs in a tissue model after delivery using SLO and cholesterol conjugation respectively. Tissues were stained with DAPI to identify nuclear compartment of the cells in a tissue. (b) Fluorescence line scan to map spatial variation in fluorescence intensity of ONs with respect to the nuclear staining. The representative cells are pointed out by white arrows in panel a. Scale bar: 50 μm.

Synergistic Combination between Microneedles and Cholesterol-Conjugated ONs for Intracellular Delivery of ONs in 3D Tissue Models. To overcome the limitation of SLO toxicity, which limits its in vivo use, delivery of cholesterol-conjugated ONs coated on microneedles was evaluated using 3D tissue models. The design of the cholesterol-conjugated ONs is shown in Table 1. Similar to the experimental approach in Figure 2, the ON distribution in the radial and axial directions of a tissue model was mapped using confocal microscopy. Imaging results in Figure 3a show that the cholesterolconjugated ONs can be delivered more than 500 μm along the radial (lateral) direction from the microneedle insertion point within 35 min of incubation post coated microneedle removal. The results also show that the fluorescence intensity of ONs does decrease with an increase in radial (lateral) distance from the microneedle insertion point and the decrease in fluorescence intensity is similar to the SLO-mediated intracellular delivery of ONs (Figure 2a). Figure 3b maps the distribution of ONs along the axial (depth) direction of a tissue model. Imaging results show that ONs can be delivered throughout the thickness of the tissue models. Figure 3c shows variation in the mean fluorescence intensity corresponding to local concentration of ONs along the axial direction of a tissue model. Similar to the results of SLO-mediated delivery (Figures 2b and 2c), the result from cholesterol-mediated delivery shows that the relative fluorescence intensity near the insertion point is higher as compared to the distal portion of the tissue model. However, the rate of decrease of the mean fluorescence intensity as a

confocal microscopy. Results of the imaging measurements are shown in Figure S2 in the Supporting Information. The results show a diffuse distribution of ONs in the extracellular matrix of a tissue model. The results also show a limited intracellular penetration of ONs. This result is in contrast to the efficient intracellular delivery of ONs in SLO treated tissue models (Figure 2). Together, the results in Figure 2 and Figure S2 in the Supporting Information demonstrate that the combination of SLO and microneedles can promote intracellular delivery of ONs in intact 3D tissue. Potential cytotoxic effects of SLO however limit the use of SLO-based delivery approaches for clinical use. The influence of cytotoxic effects of SLO is enhanced in 3D tissues as compared to 2D cell culture models as the excess SLO cannot be simply removed by a simple washing step.59,60 Figure S3 in the Supporting Information illustrates that incubation of SLO with model tissue for 48 h results in increased trypan blue staining of cells, indicating that the cellular membrane is permeabilized. The increased membrane permeability can induce cytotoxic effects in cells. For this measurement, SLO was coated on microneedles and delivered to the tissue model using the same protocol as used in Figure 2. After removal of microneedle following an initial 5 min of incubation, the tissues were incubated with delivered SLO for 48 h. Despite these limitations, the SLO-based delivery approach for the ONs can be a viable option for diagnostic applications using ex vivo tissue samples. Figure S4 in the Supporting Information further illustrates that microneedle insertion by itself does not cause any cell damage. G



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Figure 5. Distribution of cholesterol-conjugated ONs in a 3D tissue model after 24 h of ON incubation. The cholesterol-conjugated ONs were coated on microneedles and delivered to tissue models. Following removal of the microneedles, the tissue models were incubated for 24 h. Confocal images were acquired at different positions in a tissue model at an approximate distance of 1 mm from the microneedle insertion point. Scale bar represents 50 μm.

incubated for 24 h. Imaging results (Figure 5) show that, with longer incubation time (24 h), the ONs can be delivered to distances greater than 1 mm from the microneedle insertion points. These results also indicate that the variation in fluorescence intensity of ON probes as a function of distance within a tissue section was significantly reduced with longer incubation time (24 h) in contrast to 35 min of incubation (Figures 2a and 3a). Antisense-Mediated Silencing of GFP Expression in Cells of Tissue. To demonstrate functional activity of the ONs after delivery in 3D tissue models, antisense-GFP ONs (Table 1) were conjugated with cholesterol and delivered to tissue models using coated microneedles. Nucleotide sequence of this ON was selected on the basis of the results of a prior study.61 The imaging result in Figure 6a shows that, after 36 h, partial but significant reduction in the GFP expression was observed in the antisense treated tissue as compared to the control. Quantification of the mean fluorescence intensity of the tissue sections (results of three independent experiments and average of 5 tissue sections) is represented in Figure 6b. These results demonstrate that the anti-GFP ONs delivered using a synergistic combination of microneedles and cholesterol were functionally active.

function of distance was smaller for the cholesterol-conjugated ONs (Figure 3c) as compared to the same measurements for the SLO-mediated delivery of ONs (Figure 2c). Differences in Intracellular Distribution of ONs Delivered Using Cholesterol-Conjugated ONs and SLO in 3D Tissue Phantom Models. We further investigated differences between SLO and cholesterol as intracellular delivery vehicles of ONs. Figure 4 compares the intracellular distribution of ONs in 3D tissue models delivered using free SLO or cholesterol conjugation to ONs. Imaging results in Figure 4a show that SLO enables ON to localize in the nuclei and the cytoplasm of cells while cholesterol causes the ON to preferentially stay in the cytoplasm. To quantify differences in the intracellular localization of the ONs, a line scan representing variation in fluorescence intensity of the nuclear stain (DAPI) and the fluorescently labeled ONs as a function of spatial location within a representative cell (validated on the basis of the same analysis of approximately 50 cells in a tissue phantom) in a tissue model was measured. The results in Figure 4b demonstrate a significant spatial overlap between the intracellular distributions of ONs and the nuclear staining dye for the SLO-mediated delivery. In the case of the cholesterolconjugated ONs (Figure 4c), limited overlap between the intracellular distributions of cholesterol-conjugated ONs and the nuclear staining dye was observed. These results show that the cholesterol-conjugated oligonucleotides were predominantly distributed in the cytoplasm of cells while the ONs delivered using the SLO were distributed in both the cytoplasm and the nucleus of a cell. Longer Incubation Time Can Improve Intratissue Distribution of ONs in 3D Tissue Models. Because SLO is toxic to cells (Figure S3 in the Supporting Information), its long-term incubation with 3D tissue models was not evaluated. Cholesterol on the other hand is nontoxic and can be safely used for longer durations. Therefore, to evaluate influence of longer incubation time on intratissue distribution of ONs in 3D tissues, tissue models were allowed to incubate for 24 h instead of 35 min, after delivery of cholesterol-conjugated ONs using the microneedle array. The coated microneedle array by itself stays in the tissue only for 5 min to enable delivery and is not

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DISCUSSION Biochemical Conjugation Coupled with Coated Microneedles Helps Deliver ONs into Cells of Tissues. This study for the first time introduces and evaluates the potential of microneedles for widespread localized delivery of ONs in 3D tissues and their synergistic combination with biochemical approaches to successfully deliver ONs in cells situated in 3D tissues. The results of this study have demonstrated that ONs can be uniformly coated on microneedle arrays and the ON-coated microneedle arrays can efficiently and rapidly deliver the ONs in 3D tissue models. The reduction in GFP expression in HeLa cells provides demonstration of the functional effectiveness of this approach. The coating formulation used in this research was based on the FDA approved excipients,53,55−57,62 thus the overall approach H



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Furthermore, 2D arrays could also be developed to better distribute ONs over large tissue areas. We have also tested the approach in an ex vivo human oral biopsy. As seen in Figure S6 in the Supporting Information, widespread intratissue delivery of ONs can be achieved using microneedles, suggesting the clinical potential of this approach. Differences in Intracellular Localization of ONs Based on the Biochemical Approaches. Intracellular delivery of ONs in 3D tissues using the cholesterol-conjugated ON has significant advantages as compared to the SLO-mediated delivery of the ONs. These advantages result from toxicity and high MW of SLO. Due to toxicity concerns, cells, especially in vivo, cannot be incubated with SLO for an extended period of time, while the high MW of SLO (∼60 kDa) can significantly limit the diffusion of SLO molecules in 3D tissue, thus reducing uniformity and efficiency of intracellular delivery of ONs in 3D tissues. High resolution imaging of tissue models further demonstrated significant differences in intracellular localization of ONs based on the biochemical approach used. In the case of SLO-mediated delivery of the ONs, a large fraction of ONs were localized in the nuclei of cells in contrast to predominantly cytoplasmic localization of the cholesterolconjugated ONs. These results are in agreement with a prior study46 in which SLO was used for improving the nuclear localization of ONs in cells. The precise mechanism of nuclear accumulation of ON probes using SLO is not well established, although prior studies44,63,64 have observed a similar nuclear accumulation of ON probes. SLO-induced pore formation in membranes is caused by binding of SLO monomers to cholesterol rich domains on cell membranes.64,65 It is possible that, after initial pore formation on plasma membrane, excess SLO monomers can enter the cytoplasm and induce similar pore formation on nuclear and ER membranes. This effect may be more significant in this particular application as the excess SLO cannot be easily removed from the tissue model as compared to 2D cell culture model systems. In case of cholesterol-conjugated ONs, intracellular delivery is achieved by a receptor-mediated internalization of the ONs.45,46 Cholesterol-conjugated ONs have been shown to bind plasma lipoproteins. These lipoprotein complexes are then taken up by cells via specific membrane receptors, i.e., lowdensity lipoproteins (LDLs) and high-density lipoproteins (HDLs).34,66 Upon internalization, some fraction of endosomal entrapped ONs are released to the cytoplasm. The precise mechanism of ON release to the cytoplasm from endosomes is not well established,34 however, based on the biological activity of ON probes there is significant evidence to support this phenomenon. These variations in the intracellular localization can have a significant impact on functional activity of the ONs. The target cellular compartment for ONs certainly depends on where the biological target resides in the cell. Although many studies have focused on cytoplasmic activity of ONs for controlling gene expression, there are studies which have explored the potential of targeting DNA/RNA molecules into the nucleus of cells for achieving the same goals.67−72 In addition to silencing gene expression, ONs can be used for diagnostic applications such as molecular beacon based measurement of gene expression in cells. The gene expression measurements can be made in both cytoplasmic and nuclear compartments.2,73 Thus, while the cellular compartment for ON delivery will be dictated by the specific application, microneedles can still be used to achieve

Figure 6. Functional activity of microneedle delivered ON probes was evaluated based on antisense-mediated silencing of GFP expression in 3D tissue models. (a) Confocal imaging micrograph illustrating the comparison between the control and the anti-GFP treated tissue models. (b) Quantitative measurement to characterize reduction in the GFP fluorescence signal in cells within a tissue model. Scale bar represents 50 μm.

can be readily translated for potential preclinical and clinical evaluations. In addition, the influence of microneedle insertion on cell viability in tissue models was also evaluated. The results (Supporting Information, Figure S4) illustrate that microneedle insertion does not influence cell viability in 3D tissue models. These results highlight that microneedle arrays provide a minimally invasive approach to deliver ONs in tissues. Widespread Intratissue Distribution and Intracellular Delivery of ONs. To clinically utilize ONs for theranostic applications, it is important that all cells in the target tissue be exposed to the ON molecule for uptake. Typically, the extracellular matrix offers significant diffusive resistance to molecular motion, which gets further enhanced with increase in molecular size and charge. Thus, as seen in Figure S5 in the Supporting Information, topical application of the ONs only produces superficial (approximately 20 μm) penetration into the tissue. The diffusive barrier offered by the tissue to ON movement is also quite apparent from the differences in the lateral spreading of ONs in 3D tissues. By increasing the incubation period from 35 min to 24 h, the variation in fluorescence intensity of ONs in intratissue space was reduced, and the fluorescence signals were detected at larger distances (approximately 1 mm) from the microneedle insertion points. From a clinical perspective, it will be attractive if the diffusive results achieved after 24 h incubation could be obtained within a couple of hours. An important parameter that could be readily tuned to optimize the diffusive rate and coverage is the microneedle spacing. By reducing microneedle spacing, ONs could be delivered closer to each other, thus reducing the distance ONs must travel to achieve uniformity of distribution. I



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(3) Gao, Y.; Qiao, G.; Zhuo, L.; Li, N.; Liu, Y.; Tang, B. A tumor mRNA-mediated bi-photosensitizer molecular beacon as an efficient imaging and photosensitizing agent. Chem. Commun. 2011, 47 (18), 5316−5318. (4) Elsabahy, M.; Nazarali, A.; Foldvari, M. Non-viral nucleic acid delivery: key challenges and future directions. Curr. Drug Delivery 2011, 8 (3), 235−244. (5) Finotto, S.; Buerke, M.; Lingnau, K.; Schmitt, E.; Galle, P. R.; Neurath, M. F. Local administration of antisense phosphorothioate oligonucleotides to the c-kit ligand, stem cell factor, suppresses airway inflammation and IL-4 production in a murine model of asthma. J. Allergy Clin. Immunol. 2001, 107 (2), 279−286. (6) Mahato, R. I.; Cheng, K.; Guntaka, R. V. Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA. Expert Opin. Drug Delivery 2005, 2 (1), 3−28. (7) Scherer, L. J.; Rossi, J. J. Approaches for the sequence-specific knockdown of mRNA. Nat. Biotechnol. 2003, 21 (12), 1457−1465. (8) Sepp-Lorenzino, L.; Ruddy, M. Challenges and opportunities for local and systemic delivery of siRNA and antisense oligonucleotides. Clin. Pharmacol. Ther. 2008, 84 (5), 628−632. (9) Berdugo, M.; Valamanesh, F.; Andrieu, C.; et al. Delivery of antisense oligonucleotide to the cornea by iontophoresis. Antisense Nucleic Acid Drug Dev. 2003, 13 (2), 107−114. (10) Birchall, J. C.; Marichal, C.; Campbell, L.; Alwan, A.; Hadgraft, J.; Gumbleton, M. Gene expression in an intact ex-vivo skin tissue model following percutaneous delivery of cationic liposome-plasmid DNA complexes. Int. J. Pharm. 2000, 197 (1−2), 233−238. (11) Fletcher, S.; Honeyman, K.; Fall, A. M.; Harding, P. L.; Russell, J.; Wilton, S. D. Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucteotide. J. Gene Med. 2006, 8 (2), 207−216. (12) Golzio, M.; Mazzolini, L.; Moller, P.; Rols, M. P.; Teissie, J. Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery. Gene Ther. 2005, 12 (3), 246−251. (13) Phull, H.; Lien, Y. H. H.; Salkini, M. W.; Escobar, C.; Lai, L. W.; Ramakumar, S. Delivery of intercellular adhesion molecule-1 antisense oligonucleotides using a topical hydrogel tissue sealant in a murine partial nephrectomy/ischemia model. Urology 2008, 72 (3), 690−695. (14) Robinson, K. A.; Chronos, N. A.; Schieffer, E.; et al. Endoluminal local delivery of PCNA/cdc2 antisense oligonucleotides by porous balloon catheter does not affect neointima formation or vessel size in the pig coronary artery model of postangioplasty restenosis. Catheterization Cardiovasc. Diagn. 1997, 41 (3), 348−353. (15) Panyam, J.; Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Delivery Rev. 2003, 55 (3), 329−347. (16) Dong, L.; Huang, Z.; Cai, X.; et al. Localized Delivery of Antisense Oligonucleotides by Cationic Hydrogel Suppresses TNFalpha Expression and Endotoxin-Induced Osteolysis. Pharm. Res. 2011, 28 (6), 1349−1356. (17) Alexander, J. C.; Pandit, A.; Bao, G.; Connolly, D.; Rochev, Y. Monitoring mRNA in living cells in a 3D in vitro model using TATpeptide linked molecular beacons. Lab Chip 2011, 11 (22), 3908− 3914. (18) Dokka, S.; Cooper, S. R.; Kelly, S.; Hardee, G. E.; Karras, J. G. Dermal delivery of topically applied oligonucleotides via follicular transport in mouse skin. J. Invest. Dermatol. 2005, 124 (5), 971−975. (19) Kigasawa, K.; Kajimoto, K.; Nakamura, T. Noninvasive and efficient transdermal delivery of CpG-oligodeoxynucleotide for cancer immunotherapy. J. Controlled Release 2011, 150 (3), 256−265. (20) Moschos, S. A.; Frick, M.; Taylor, B. Uptake, Efficacy, and Systemic Distribution of Naked, Inhaled Short Interfering RNA (siRNA) and Locked Nucleic Acid (LNA) Antisense. Mol. Ther. 2011, 19 (12), 2163−2168. (21) Lai, S. Y.; Koppikar, P.; Thomas, S. M.; et al. Intratumoral Epidermal Growth Factor Receptor Antisense DNA Therapy in Head and Neck Cancer: First Human Application and Potential Antitumor Mechanisms. J. Clin. Oncol. 2009, 27 (8), 1235−1242.

the desired targeting by combining them with the appropriate biochemical intracellular targeting modality.

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CONCLUSIONS This study for the first time examined the potential of delivering ONs into cells of 3D tissues by combining microneedle-based intratissue delivery with biochemical approaches to guide the ONs further into cells of the 3D tissue. Microneedles were uniformly coated with ONs. Delivery efficiency of ONs coated on microneedle surfaces into 3D tissue models was greater than 90%. Overall this study shows that synergistic use of microneedles and SLO or cholesterol conjugation of ONs results in widespread and efficient intracellular delivery of ONs in 3D tissue models. Co-insertion of microneedles coated with ONs and SLO into 3D tissue models resulted in delivery of ONs into both the cytoplasm and nucleus of cells. Within a short incubation time (35 min), ONs were observed both laterally and along the depth of a 3D tissue up to a distance of 500 μm from the microneedle insertion point. Similar intratissue distribution of ONs was achieved upon delivery of ON−cholesterol conjugates. The intratissue spread of ON probes with respect to microneedle insertion points improved with longer incubation times (24 h). Using cholesterol-conjugated ONs, delivery of ON probes was limited to the cytoplasm of cells within a tissue. Finally, delivery of a cholesterol-conjugated anti-GFP ON resulted in reduction of GFP expression in HeLa cells. In summary, the results of this study provide a novel approach for efficient intracellular delivery of ONs in tissues. The results of this study can have a significant impact on development of ON-based therapeutic and diagnostic applications for localized tissues.

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details and figures including micrographs of a control tissue phantom, delivery of microneedle coated ONs in a 3D tissue model without SLO, cytotoxicity of SLO on tissue phantom, influence of microneedle insertion on cell viability in a tissue model, comparison between topical application and microneedle-based delivery of ON probes in 3D tissue phantoms, and intratissue distribution of ON probes in human oral biopsies. 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]; [email protected]. Author Contributions ‡

Equal contribution.

Notes

The authors declare the following competing financial interest(s): H.S.G. is an inventor of a patent that has been licensed to a company developing microneedle-based products. This potential conflict of interest has been disclosed and is managed by Texas Tech University.

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Microprecision Delivery of Oligonucleotides in a 3D Tissue Model and

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