Nanodiamond Vectors Functionalized with Polyethylenimine for

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Nanodiamond Vectors Functionalized with Polyethylenimine for siRNA Delivery Mark Chen,† Xue-Qing Zhang,† Han B. Man,† Robert Lam,† Edward K. Chow,‡ and Dean Ho*,† †

Department of Biomedical Engineering and Department of Mechanical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States, and ‡G.W. Hooper Foundation, University of California San Francisco, HSW 1501, San Francisco, California 94143, United States

ABSTRACT The enormous therapeutic potential of RNA interference (RNAi) has long been recognized. While efficient small interfering RNA (siRNA) delivery vectors exist, many sacrifice biocompatibility, which can challenge their applicability as a therapeutic agent. Nanodiamonds (NDs) represent promising strategies for efficient siRNA delivery given the multitude of beneficial properties integrated into one platform that include uniform particle sizes, material scalability, the ability to carry nearly any type of therapeutic, and preserved biocompatibility, among others. Here we present a broadly applicable ND delivery platform that demonstrates biocompatible siRNA delivery with enhanced efficacy in media, signifying the translational potential of this approach. SECTION Nanoparticles and Nanostructures

ince the discovery of RNA interference (RNAi)1 by Fire and Mello, researchers have recognized its incredible therapeutic potential. Specifically, much of the research geared toward realizing the promise of RNAi therapy has focused on nanoparticles. A wide spectrum of nanoparticles have been explored for applications in medicine with demonstrated potential.2-5 In the present work we have developed a hybrid nanodiamond-polyethyleneimine (ND-PEI) material as a broadly applicable platform for small interfering RNA (siRNA) delivery. Most importantly, we have also considered the delivery efficiency of our vector in vitro under translationally relevant conditions. Specifically, under biological conditions in media containing serum, ND-PEI exhibited greater transfection efficiency than Lipofectamine, which is a gold standard for transfection. Nanodiamonds (NDs) have been explored for ND-protein interactions6 and imaging.2-10 Additionally, we have previously demonstrated the utility of NDs as a drug delivery platform for an extensive array of drugs.5,11 Most recently, we have demonstrated additional applications of NDs as platforms for gene and therapeutic protein delivery.12-14 Given their innate biocompatibility, scalability, water-solubility, functional carbon surface, and high surface area-to-volume ratio, NDs are a promising platform for designing an siRNA delivery vector.15,16 Consequently, based on our observations in using NDs for gene delivery, we hypothesized that the versatility of the platform may extend to siRNAs as a delivery vector as well. The preparation of ND-PEI siRNA complexes was accomplished by coating NDs with 800 Da PEI, and incubating with siRNAdirectly before transfection (Scheme 1). Several washes were performed to remove excess PEI. While 800 Da PEI is a less efficient transfection reagent due to its low molecular

weight (LMW), high molecular weight (HMW) PEI sacrifices biocompatibility for better transfection efficiency.17 Additionally, research has shown cross-linked LMW PEI increases its transfection effciency.18 Our approach utilizes the carboxylated ND surface as a platform for LMW PEI. The binding properties of PEI to ND and siRNA are well established.13,19-21 Essentially, the interactions are electrostatic owing to the opposing surface charge of NDs and PEI, and PEI and siRNA. These differences in charge are what require the combination of ND and PEI to reconcile and allow successful complexing between ND, PEI, and siRNA. Without PEI, siRNA loading on NDs would not be significant. Additionally, without NDs, LMW PEI transfection efficiency remains poor. Therefore, we hypothesized that this combination of materials would not only possess the innate biocompatibility inherent to both 800 Da PEI and ND, but also act as a platform for larger molecular weight complexes to improve transfection efficiency. To confirm the presence of PEI on the NDs, dynamic light scattering (DLS) was performed on the samples. The ζ potential measurements show a net positive charge on the final ND-PEI complexes (Figure 1). Furthermore, a clear interaction between the siRNA and ND-PEI complexes is evidenced by the changes in particle size as the charges on the siRNA and ND-PEI neutralize, then build up to a positive potential. Gel electrophoresis was also performed to confirm siRNA binding with ND-PEI, which revealed complete loading at a w/w ratio of 1:5 siRNA to ND (Scheme 1). The binding gel

S

r 2010 American Chemical Society

Received Date: September 23, 2010 Accepted Date: October 14, 2010 Published on Web Date: October 19, 2010

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DOI: 10.1021/jz1013278 |J. Phys. Chem. Lett. 2010, 1, 3167–3171

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Scheme 1. Preparation of ND-PEI siRNA and Complexing Ratios

Complexing ratios are based on a siRNA to ND-PEI w/w ratio.

that, despite internalization, the NDs may exhibit an siRNA sequestering/slow release effect similar to other studies with small molecules5 compared to a liposomal vector such as Lipofectamine, which had roughly the same percentage of internalization at 98.8%, but 44.9% GFP knockdown (Figure S2). The exact release mechanism remains an ongoing area of investigation. Although the optimal binding ratio of siRNA: ND-PEI was achieved at 1:5, this does not demonstrate that it is the optimal ratio for transfection. Thus, we chose w/w ratios from 1:3 to 1:20 to determine the best loading for transfection. To do this we performed transfections in 96-well plates and looked for the presence of GFP after cell lysis (Figure 3a). The data was normalized to total protein content per well determined via bicinchoninic acid (BCA) assay. Additionally, we used the same w/w ratios for siRNA-particle complexes using either only ND or only PEI to demonstrate that the delivery properties of the ND-PEI are unique to the hybrid material and not either material independently. GFP fluorescence readings reveal knockdown to be greatest in cells treated with Lipofectamine þ siRNA (Figure 3a). The 1:3 ratio ND-PEI þ siRNA exhibits the next highest efficiency, knocking down roughly half as much GFP as the Lipofectamine. Relative to the control, both Lipofectamine and ND-PEI demonstrate a significant decrease of 75.6% and 62.2% in GFP expression, respectively. Interestingly, the ND-PEI performed much better than its standalone ND and PEI counterparts. Furthermore, we performed all transfections using Dulbecco's modified Eagle's medium (DMEM) with 10% serum and 1% antibiotics except those marked TM for reduced-serum transfection media. It is important to note that for transfections involving serum proteins, ND-PEI actually performed better than Lipofectamine (Figure 3a). This observation attests to the potential utility of ND-PEI in an in vivo system that is more closely represented by DMEM with serum. These data are in agreement with our hypothesis that the ND platform would enhance the transfection efficiency of LMW PEI, as also demonstrated by other studies.18 Most importantly, it demonstrates that the ND-PEI material possesses properties exclusive to the hybrid material, rather than each individual material.

Figure 1. ND-PEI siRNA characterization via DLS: (a) particle size; (b) ζ potential.

was run as a standard experiment to determine the weight ratio where ND could load siRNA with no excess.21,22 Initial knockdown of green fluorescence protein (GFP) using ND-PEI siRNA was confirmed with confocal imaging (Figure 2a,b and Supporting Information, Figure S1 ). The ability for ND-PEI particles to internalize into cells with siRNA was investigated using cyanine-5 (Cy5)-labeled siRNA and fluorescence-activated cell sorting (FACS) (Figures 2c and S2). At a ratio of 1:3 siRNA:ND-PEI, the internalization was 97.1% Interestingly, the GFP knockdown was 23.3% This suggests


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Figure 2. Confocal and FACS of GFP knockdown in M4A4þGFP cells: (a) negative control; (b) ND-PEI siRNA; (c) GFP knockdown.

only gene delivery, but also siRNA delivery. Given the scalability inherent in the facile one-step production of the complexes, ND-PEI may be a promising platform for therapeutic applications.

EXPERIMENTAL METHODS ND-PEI Complex Preparation. ND gel (15% w/v in water) was obtained from the NanoCarbon Research Institute, Ltd., Japan. Anti-GFP siRNA was obtained from Ambion: Sense 50 CAA GCU GAC CCU GAA GUU Ctt-30 ; Antisense 50 -GAA CUU CAG GGU CAG CUU Gtt-30 . All commercial reagents were used without purification. ND gel was diluted in pure water at the desired concentration and sonicated with a Branson 2510 sonicator for 2 h. To prepare ND-PEI, the ND solution was mixed with 800 Da PEI (Sigma-Aldrich) at a 1:20 ratio of excess 800 Da PEI. Following one minute of vortexing, the sample was centrifuged for 2 h at 14000 rpm to pellet ND-PEI complexes. The excess PEI was removed with the supernatant, and the pellet was resuspended in pure water. Three washes in total were performed to remove excess PEI. The final product was an optically clear solution. Prior to usage, siRNA was incubated with ND-PEI and added directly to the experiment within 10 min. Particle Size and Potential Measurements. Characterization of ND-PEI complexes was performed via DLS. ND-PEI complexes with siRNA were prepared in 1 mL of pure water at various siRNA:ND-PEI weight ratios ranging from 0 to 1:20. siRNA and ND-PEI were allowed to complex for 10 min. Measurements were performed with a Zetasizer Nano ZS (Malvern). Size measurements were performed at 25 °C at a 173° scattering angle. Mean diameter was determined via cumulative analysis. ζ potential measurements were performed using folded capillary cells at 25 °C. Retardation Gel. FlashGel (Lonza) was used for electrophoresis of siRNA-nanoparticle complexes. siRNA:ND-PEI weight ratios ranging from 1:20 to 20:1 were run on the gel at 200 V for 2 min. Gels were imaged with the FlashGel system. The experiment was repeated in triplicate. Cell Culture. GFP-expressing M4A4 breast cancer cells (ATCC) were grown in DMEM, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Reducedserum transfection media was used for all positive control experiments. Cells were maintained at 37 °C in 5% CO2. Cells were seeded in 96-well plates at a density of 10 000 cells per well and allowed to grow for 1 day prior to transfection for gene knockdown analysis. The media was supplemented with fresh media 5 h after transfection. The wells were aspirated and replaced with fresh media 24 h after transfection.

Figure 3. (a) Fluorescence readings of GFP knockdown; RLU = relative light units. (b) MTT cell viability assay.

After demonstrating that ND-PEI serves as an effective delivery agent of siRNA, we examined the biocompatibility of ND-PEI siRNA complexes via MTTassay. This is important, as liposomal siRNA delivery, while effective, is often associated with enhanced cytotoxicity. As shown in Figure 3b, Lipofectamine results in 26.4% cytotoxicity, while ND-PEI has 96.8% viability;close to zero cytotoxicity. These data demonstrate that ND-PEI is both a highly effective delivery platform for siRNA and biocompatible. The importance of biocompatibility for potential siRNA delivery platforms such as ND-PEI cannot be overstated, as safety is of paramount importance for future in vivo applications. By combining two biocompatible materials;NDs and LMW PEI;we have successfully created a mimic of an HMW PEI vector without the cytotoxicity and achieving nearly the same efficiency. This work introduces the concept of siRNA delivery via an ND-based platform and further substantiates the broad applicability of NDs as a platform for not


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Cells were harvested 48 h after transfection at 70-80% confluence. All experiments used cells with no more than eight passages. Confocal Imaging. One 105 cells were seeded in clear 0.17 mm thick Delta T culture dishes (Bioptechs) and allowed to grow overnight before transfection with ND-PEI siRNA complexes. After 48 h, cells were imaged on a Zeiss Confocal laser scanning microscope (Carl Zeiss LSM510) with a C-Apochromat 40/1.20 W korr water immersion objective. Cells were maintained at 37 °C with a Delta T5 μ-ENVIRONMENTAL Culture Dish Controller (Bioptechs) during observation. Bright field images were performed via differential interference contrast (DIC) microscopy. Images were converted into TIFF format with ZEN 2009 software (Carl Zeiss) and processed through Volocity software (Perkin-Elmer). Adobe Photoshop was used to normalize the background of the ND-PEI siRNA overlay image to the negative control for accurate comparison. Gene knockdown. siRNA-nanoparticle complexes at 1:20, 1:10, 1:15, and 1:3 weight ratios were prepared as described above. siRNA-nanoparticle complexes were transfected into each well to a final concentration of 60 nM siRNA for 20 000 cells in a 96-well plate. Cells were allowed to grow for 48 h before analysis and harvested with RIPA buffer for complete cell lysis. The plates were then immediately analyzed for GFP using 485 nm excitation and 528 nm emission wavelengths with a Biotek Synergy4 microplate reader. The total protein content of each well was them determined via BCA assay (Pierce) per the manufacturer's instructions. GFP readings were normalized to the total protein content per well. The experiment was performed in triplicate, n = 4 per experiment. FACS. Cell seeding and transfection was performed as described above, except using 50 -Cy5-labeled anti-GFP siRNA (Qiagen). Forty-eight hours after transfection, the wells were aspirated and washed with warm phosphate buffered saline (PBS). Trypsin-EDTA was added to each well to detach adherent cells. The cells were resuspended in warm PBS supplemented with 1% calf bovine serum before flow cytometry. Cellular internalization of nanoparticles was analyzed with the BD LSR II (BD Biosciences) flow cytometer for 1500 events per sample. Data were analyzed with BD FACSDiVa software. Quadrants markers and voltages were set using controls. MTT Cell Viability Assay. Reverse transfection of siRNAnanoparticle complexes was performed in 96-well plates seeded at 15 000 cells per well to a final siRNA concentration of 60 nM. The cells were allowed to grow for 48 h before performing the assay. The MTT assay (Invitrogen) was performed according to the manufacturer's instructions. The experiment was performed in triplicate, n = 3 per experiment.

ACKNOWLEDGMENT D.H. gratefully acknowledges support from

a National Science Foundation CAREER Award (CMMI-0846323), National Science Foundation Mechanics of Materials program grant (CMMI-0856492), V Foundation for Cancer Research V Scholars Award, National Science Foundation Center for Scalable and Integrated NanoManufacturing (SINAM) Grant DMI-0327077, and Wallace H. Coulter Foundation Early Career Award in Translational Research. E.K.C. gratefully acknowledges support from the American Cancer Society. We thank the O'Halloran group, Northwestern University, for confocal imaging facility access.

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SUPPORTING INFORMATION AVAILABLE Supplementary

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figures of confocal cellular imagery and flow cytometry analysis. This material is available free of charge via the Internet at http:// pubs.acs.org (13)

AUTHOR INFORMATION Corresponding Author:

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*To whom correspondence should be addressed. E-mail: d-ho@ northwestern.edu.


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